Replication deficient adenoviral TNF vector

ABSTRACT

An adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, (b) a nucleic acid sequence coding for TNF, and (c) a radiation inducible promoter operably linked to the nucleic acid sequence coding for TNF. This invention also provides an adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, (b) a nucleic acid sequence coding for TNF, and (c) a spacer element of at least 15 base pairs in the E4 region of the adenoviral genome. A method of producing an adenoviral vector and a method of treating a tumor or cancer in a host comprising administering an anti-cancer or anti-tumor effective amount of the adenoviral vector of the present invention also is provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This patent application is a divisional of U.S. patent application Ser. No. 09/604,694, filed Jun. 27, 2000.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention pertains to a replication deficient adenoviral vector comprising a nucleic acid sequence coding for tumor necrosis factor (TNF), as well as a method of constructing and using such vector.

BACKGROUND OF THE INVENTION

[0003] Tumor necrosis factor (TNF), especially TNF-α, is well-known for its anti-tumor effects and ability to act synergistically with radiation therapy. For example, certain replication deficient adenoviral vectors comprising the TNF-α gene have been used in conjunction with radiation therapy to treat tumors in animals with some success (e.g., Hallahan et al., Nat. Med., 1, 786-91 (1995)). The use of TNF as an anti-cancer/anti-tumor agent, however, has been limited by its severe systemic effects.

[0004] There remains a need for replication deficient TNF adenoviral vectors that have greater flexibility in their construction and use, and can provide greater success in the treatment of a tumor or cancer. The present invention provides such a vector, as well as a method of constructing such vector, and a therapeutic method involving the use of such vector.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides an adenoviral vector comprising (a) an adenoviral genome deficient in the early growth-4 (E4) region of the adenoviral genome, (b) a nucleic acid sequence coding for TNF, and (c) a radiation inducible promoter operably linked to the nucleic acid sequence coding for TNF. This invention also provides an adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, (b) a nucleic acid sequence coding for TNF, and (c) a spacer element of at least 15 base pairs in the E4 region of the adenoviral genome.

[0006] A method of producing an adenoviral vector also is provided by the present invention comprising (a) providing an adenoviral genome that is deficient in the E4 region of the adenoviral genome, (b) inserting a nucleic acid sequence coding for TNF into the adenoviral genome, and (c) inserting a radiation-inducible promoter into the adenoviral genome such that it is operably linked to the nucleic acid sequence coding for TNF. This invention further provides a method of treating a tumor or cancer in a host comprising administering an anti-cancer or anti-tumor effective amount of the adenoviral vector of the present invention to a host in need thereof.

[0007] The present invention may best be understood with reference to the accompanying drawings and in the following detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1A is a schematic representation of a vector containing an unmodified adenoviral genome.

[0009]FIG. 1B is a schematic representation of a vector containing a modified adenoviral genome and the genetic elements in accordance with the present invention.

[0010]FIG. 2 is a plot of tumor volume against time for a tumor treated in a variety of manners, including with the adenoviral vector of the present invention.

[0011]FIG. 3 is another plot of tumor volume against time for a tumor treated in a variety of manners, including with the adenoviral vector of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides an adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, (b) a nucleic acid sequence coding for TNF, and (c) a radiation inducible promoter operably linked to the nucleic acid sequence coding for TNF. The present invention also provides an adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, (b) a nucleic acid sequence coding for TNF, and (c) a spacer element of at least 15 base pairs in the E4 region of the adenoviral genome.

Adenoviral Genome

[0013] Any subtype, mixture of subtypes, or chimeric adenovirus can be used as the source of the viral genome for generation of an adenoviral vector in conjunction with the present invention. Preferably the genome of a human serotype adenovirus is used, such as a type 2 (Ad2) or type 5 (Ad5) adenoviral genome. Although any suitable adenoviral genome can be used in conjunction with the present invention, the Ad5 adenoviral genome is most preferred, and the present invention is described further herein with respect to the Ad5serotype.

[0014] The adenoviral genome used in conjunction with the present invention is desirably replication deficient. A deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion of sufficient genetic material of the viral genome to impiar or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part and to provide room in, or capacity of, the viral genome for the insertion of a nucleic acid sequence that is foreign to the viral genome. Such a deficiency can be in a gene or genome region essential or unessential for propagation of the adenoviral vector in a non-complementing cellular host. A deficiency in an adenoviral genome region essential for such propagation (e.g., early region 1 (E1), early region 2A (E2A), early region 2B (E2B), early region 4 (E4), late region 1 (L1), late region 2 (L2), late region 3 (L3), late region 4 (L4), and late region 5 (L5)) renders an adenoviral vector based on that adenoviral genome replication deficient.

[0015] The adenoviral vector of the present invention desirably is multiply replication deficient, i.e., it is deficient in at least two genome regions required for viral propagation in a non-complementing cellular host (i.e., viral replication in vitro). Such regions include the E1, E2, E4, or L1-L5 regions. Even though the E1 region can be considered as consisting of early region 1A (E1A) and early region 1B (E1B), a deficiency in either or both of the E1A and/or E1 B regions is considered as a single deficiency in the context of the present invention. In addition, such a vector can be deficient in one or more regions that are not required for viral propagation, e.g., the vectors can be additionally deficient in early region 3 (E3).

[0016] The present inventive adenoviral vector desirably is deficient in the E1 and E4 regions, preferably with the entire coding region of the E4 region having been deleted from the adenoviral vector. In the context of the present invention, the coding region refers to the portion or portions of a genomic region that encode a protein product (e.g., the open reading frames (ORFs) of the E4 region). Thus, a vector in which the entire coding region of the E4 region of the adenoviral genome has been deleted is lacking all of the ORFs of that region. The E4 region of the present inventive adenoviral vector preferably retains the native E4 promoter, polyadenylation sequence, and/or the right-side inverted terminal repeat (ITR).

[0017] The adenoviral vector of the present invention also can be deficient in one or more additional regions required for viral propagation (especially other early regions required for viral propagation, such as the E2A and/or E2B regions). More preferably, the E3 region of the adenoviral genome also is removed. Thus, preferred configurations of the present inventive adenoviral vector include (a) E1⁻ E4⁻, (b) E1⁻ E2A⁻ E4⁻, and (c) E2A⁻ E4⁻ adenoviral vectors, any of which also can be E3⁻.

[0018]FIG. 1A provides a schematic representation of the Ad5 genome. FIG. 1B is a schematic representation of an exemplary adenoviral vector, which figure shows, by comparison to the unaltered Ad5 adenoviral genome represented by FIG. 1A, that (i) the E1region (10) has been replaced by a radiation inducible promoter (14), a nucleic acid sequence encoding TNF (15), and a polyadenylation sequence (16), (ii) the E3 region (11) has been deleted (17), and (iii) the coding region of the E4 region (12) has been replaced by a spacer element (18), thereby creating a vector deficient in the E1, E3, and E4 regions.

[0019] If the adenoviral vector of the present invention is deficient in the E2A region, the vector preferably further comprises a portion of the E2A region of the adenoviral genome in the E2A deficient region, which is less than about 230 base pairs in length. Generally, the E2A region of the adenovirus codes for DBP (DNA binding protein), a polypeptide required for DNA replication. DBP is composed of 473 to 529 amino acids depending on the viral serotype. It is believed that DBP is an asymmetric protein that exists as a prolate ellipsoid consisting of a globular Ct with an extended Nt domain. Studies indicate that the Ct domain is responsible for DBP's ability to bind to nucleic acids, bind to zinc, and function in DNA synthesis at the level of DNA chain elongation. However, the Nt domain is believed to function in late gene expression at both transcriptional and post-transcriptional levels, is responsible for efficient nuclear localization of the protein, and also may be involved in enhancement of its own expression. Deletions in the Nt domain between amino acids 2 to 38 have indicated that this region is important for DBP function (Brough et al., Virology, 196, 269-281 (1993)). While deletions in the E2A region coding for the Ct region of the DBP have no effect on viral propagation, deletions in the E2A region which code for amino acids 2 to 38 of the Nt domain of the DBP impair viral propagation. Therefore, it is preferable that any multiply replication deficient adenoviral vector contain this portion of the E2A region of the adenoviral genome.

[0020] In particular, for example, the desired portion of the E2A region to be retained is that portion of the E2A region of the adenoviral genome which is defined by the 5′ end of the E2A region, specifically, positions Ad5(23816) to Ad5(24032) of the E2A region of the adenoviral genome of serotype Ad5. This portion of the adenoviral genome desirably is included in the adenoviral vector because it is not complemented in current E2A cell lines, and in its absence the requisite levels of viral propagation and fiber expression cannot be obtained in complementing cell lines.

[0021] The present invention is not limited to adenoviral vectors that are deficient in gene functions only in the early region of the genome. Also included are adenoviral vectors that are deficient in the early and late regions of the genome, as well as vectors in which essentially the entire genome has been removed, in which case it is preferred that at least either the viral ITRs and some of the promoters or the viral ITRs and a packaging signal are left intact. One of ordinary skill in the art will appreciate that the larger the region of the adenoviral genome that is removed, the larger the piece of exogenous nucleic acid sequence that can be inserted into the genome. For example, given that the adenoviral genome is 36 kb, by leaving the viral ITRs and some of the promoters intact, the capacity of the adenovirus is approximately 35 kb. Alternatively, one could generate a multiply deficient adenoviral vector that contains only the ITR and a packaging signal. This could then effectively allow for expression of 37-38 kb of foreign nucleic acid sequence from this vector. Of course, the inclusion of a spacer element in any or all of the deficient adenoviral regions will decrease the capacity of the adenoviral vector in size corresponding with the size of the spacer element sequence.

Nucleic Acid Sequence Encoding TNF

[0022] Preferably, the vector comprises at least one expression cassette which includes (i.e., comprises) a nucleic acid sequence coding for TNF. Nucleic acid sequences encoding TNF include nucleic acid sequences encoding any member of the TNF family of proteins. The adenoviral vector of the present invention preferably comprises a nucleic acid sequence coding for TNF-α. A nucleic acid sequence coding for TNF is described in detail in U.S. Pat. No. 4,879,226, which discloses a nucleic acid sequence encoding a “human TNF, in mature form, secreted from host cells” (see column 7, lines 26-28), as set forth in SEQ ID NO: 2.

[0023] The nucleic acid sequence coding for TNF is preferably located in the E1 region (e.g., replaces the E1 region in whole or in part, preferably in whole) of the adenoviral genome. For example, the E1 region can be replaced by a promoter-variable expression cassette comprising a nucleic acid seqence encoding TNF. The term “expression cassette,” as used herein, refers to any nucleic acid sequence that can be inserted into the adenoviral genome to produce a foreign gene product. For example, as shown in FIGS. 1A and 1B, the E1region (10) can be replaced by an expression cassette (13) comprising a radiation inducible promoter (14), a nucleic acid sequence encoding TNF (15), and a polyadenylation sequence (16). In addition to the expression cassette comprising the nucleic acid sequence encoding TNF, the vector can comprise other expression cassettes containing nucleic acid sequences encoding other products, which cassettes can replace any of the deleted regions of the adenoviral genome. The insertion of an expression cassette into the adenoviral genome (e.g., the E1 region of the genome) can be facilitated by known methods, for example, by the introduction of a unique restriction site at a given position of the adenoviral genome.

[0024] Preferably, the nucleic acid sequence encoding TNF further comprises a transcription-terminating region such as a polyadenylation sequence located 3′ of the region encoding TNF. Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus). A preferred polyadenylation sequence is the SV40 (Human Sarcoma Virus-40) polyadenylation sequence.

[0025] Preferably, the nucleic acid sequence encoding TNF is operably linked to (i.e., under the transcriptional control of) one or more promoter and/or enhancer elements, for example, as part of a promoter variable expression cassette. Techniques for operably linking sequences together are well known in the art. Any suitable promoter or enhancer sequence can be used in conjunction with the present invention. Suitable promoters and enhancer sequences are generally known in the art. Preferred vectors according to the present invention comprise a radiation-inducible promoter operably linked to a nucleic acid sequence encoding TNF. The use of a radiation inducible promoter provides control over transcription of the foreign gene product, for example, by the administration of radiation to a cell or host comprising the adenoviral vector. Any suitable radiation inducible promoter can be used in conjunction with the present invention. Suitable radiation inducible promoters are generally known in the art. A preferred radiation inducible promoter for use in conjunction with the present invention is the early growth region-1(Egr-1) promoter, specifically the CArG domain of the Egr-1promoter. The Egr-1 promoter has been described in detail in U.S. Pat. No. 5,206,152 and International Patent Application WO 94/06916. The promoter can be introduced into the foreign genome by methods known in the art, for example, by the introduction of a unique restriction site at a give region of the genome. Alternatively, the promoter can be inserted as part of the expression cassette comprising the nucleic acid sequence coding for TNF.

Spacer Element

[0026] The present inventive adenoviral vector, when multiply replication deficient, preferably includes a spacer element to provide viral growth in a complementing cell line similar to that achieved by singly replication deficient adenoviral vectors, particularly a singly replication deficient E1⁻ adenoviral vector. In the absence of a spacer, production of fiber protein and/or viral growth of the multiply replication deficient adenoviral vector is reduced by comparison to that of a singly replication deficient adenoviral vector. However, inclusion of the spacer in at least one of the deficient adenoviral regions, preferably the E4 region as shown in FIG. 1B (18), counteracts this defect in growth and fiber expression.

[0027] In the preferred E4⁻ adenoviral vector of the present invention wherein the L5 fiber region is retained, the spacer is desirably located between the L5 fiber region and the right-side ITR. More preferably, in such an adenoviral vector, the E4 polyadenylation sequence alone or, most preferably, in combination with another sequence, exists between the L5 fiber region and the right-side ITR, so as to sufficiently separate the retained L5 fiber region from the right-side ITR, such that viral production of such a vector approaches that of a singly replication deficient adenoviral vector, particularly a singly replication deficient E1⁻ adenoviral vector. The use of a spacer in an adenoviral vector is described in U.S. Pat. No. 5,851,806.

[0028] As the function of the replication deficient region of the genome can be provided by a complementing cell line, the spacer element does not need to provide the deficient function and can be any sequence. Thus, the spacer element is limited only by the size of the insert that the vector will accommodate. The spacer element can be of any suitable size, desirably at least about 15 base pairs (e.g., between about 15 base pairs and about 12,000 base pairs), preferably about 100 base pairs to about 10,000 base pairs, more preferably about 500 base pairs to about 8,000 base pairs, even more preferably about 1,500 base pairs to about 6,000 base pairs, and most preferably about 2,000 to about 3,000 base pairs.

[0029] The spacer element can contain any sequence or sequences which are of the desired length. The spacer element sequence can be coding or non-coding and native or non-native with respect to the adenoviral genome, but does not restore the replication function to the deficient region. The spacer element can also contain a promoter-variable expression cassette. More preferably, the spacer element comprises an additional polyadenylation sequence and/or a foreign gene. Preferably, in the case of a spacer element inserted into a region deficient for E4, both the E4 polyadenylation sequence and the E4 promoter of the adenoviral genome or any other (cellular or viral) promoter remain in the vector. In such an embodiment, the spacer element is located between the E4 polyadenylation site and the E4 promoter, or, if the E4 promotor is not present in the vector, the spacer element is proximal to the right-side ITR.

[0030] The spacer element can comprise any suitable polyadenylation sequence. Examples of suitable polyadenylation sequences include synthetic optimized sequences, as well as the polyadenylation sequences of BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus). Preferably, particularly in the E4 deficient region, the spacer element includes an SV40 (Human Sarcoma Virus-40) polyadenylation sequence. The SV40 polyadenylation sequence allows for higher virus production levels of multiply replication deficient adenoviral vectors.

[0031] A foreign gene also can function as the spacer element in the E4 deficient region of the adenoviral genome. The foreign gene is limited only by the size of the fragment the vector can accommodate and can be any suitable gene. Examples of suitable foreign genes include marker gene sequences such as pGUS, secretory alkaline phosphatase, luciferase, B-galactosidase, and human anti-trypsin; therapeutic genes; potential immune modifiers such as B3-19K, E3-14.7, ICP47, fas ligand gene, and CTLA4 genes; biologically inactive sequences (e.g. sequences that will not be transcribed to produce a product or which encode a defective or biologically inactive product), and other innocuous sequences such as the glucuronidase gene.

Vector Construction

[0032] The present invention provides a method of producing an adenoviral vector comprising (a) providing an adenoviral genome that is deficient in the E4 region of the adenoviral genome, (b) inserting a nucleic acid sequence coding for TNF into the adenoviral genome, and (c) inserting a radiation-inducible promoter into the adenoviral genome such that it is operably linked to the nucleic acid sequence coding for TNF. The present invention also provides a method of producing an adenoviral vector comprising (a) providing an adenoviral genome that is deficient in the E4 region of the adenoviral genome, (b) inserting a nucleic acid sequence coding for TNF into the adenoviral genome, and (c) inserting a spacer element into the E4 region of the adenoviral genome. As those of ordinary skill in the art will appreciate, the method provided by the present invention can include other steps or elements, such as the insertion of other nucleic acid sequences into, or deletion of such sequences from, the adenoviral genome used to provide the adenoviral vector. Furthermore, the various aspects of the present inventive method (e.g., the adenoviral genome, nucleic acid sequences coding for TNF, radiation inducible promoter, spacer element, etc.) are as previously described herein with respect to the adenoviral vector of the present invention.

[0033] The present inventive method of producing an adenoviral vector can be carried out using techniques known to those of ordinary skill in the art. In general, virus vector construction relies on the high level of recombination between adenoviral nucleic acid sequences in a cell. Two or three separate adenoviral nucleic acid sequences (e.g., DNA fragments), containing regions of similarity (or overlap) between sequences and constituting the entire length of the genome, are transfected into a cell. The host cell's recombination machinery constructs a full-length viral vector genome by recombining the aforementioned seqences. Other suitable procedures for constructing viruses containing alterations in various single regions have been previously described (Berkner et al., Nucleic Acids Res., 12, 925-941 (1984); Berkner et al., Nucleic Acids Res., 11, 6003-6020 (1983); Brough et al., Virol., 190, 624-634 (1992)) and can be used to construct multiply deficient viruses; yet other suitable procedures include, for example, in vitro recombination and ligation.

[0034] A preferred method of constructing the present inventive adenoviral vector first involves constructing the necessary deletions or modifications (such as adding a spacer element to a deleted region) of a particular region of the adenoviral genome. Such modifications can be performed, for example, in a plasmid cassette using standard molecular biological techniques. The altered nucleic acid sequence (containing the deletion or modification) then is moved into a much larger plasmid that contains up to one-half of the adenovirus genome to provide a base plasmid comprising the modified adenoviral genome. The next step is to insert an expression cassette into a desired region of the modified adenoviral genome. The expression cassette can be provided by standard methods known in the art, for example, by isolating the cassette from a plasmid. The isolated cassette then can be transfected with the plasmid DNA (containing the modified adenoviral genome) into a recipient cell. The plasmid is, optionally, linearized prior to transfection by digestion with a suitable restriction enzyme to facilitate the insertion of the expression cassette at a desired position in the adenoviral genome. Selection of a suitable restriction enzyme is well within the skill of the ordinary artisan. The two pieces of DNA recombine to form a plasmid comprising the modified adenoviral genome and the expression cassette. The plasmid is isolated from the host cell and introduced into recipient cell that complements for the missing viral functions of the recombined viral genome to produce the adenoviral vector comprising the modified viral genome and the expression cassette. The vector can be further modified by alteration of the ITR and/or packaging signal.

Complementing Cell Lines

[0035] Complementing cell lines for propagation or growth of the present inventive replication deficient adenoviral vectors are known and described in detail in U.S. Pat. No. 5,851,806 and Brough et al., Virol., 70, 6497-6501 (1996). The preferred cell lines are characterized in complementing for at least one gene function of the gene functions comprising the E1, E2, and E4 regions of the adenoviral genome. Other cell lines include those that complement adenoviral vectors that are deficient in at least one gene function from the gene functions comprising the late regions, those that complement for a combination of early and late gene functions, and those that complement for all adenoviral functions. One of ordinary skill in the art will appreciate that the cell line of choice is one that specifically complements for those functions that are missing from the recombinant replication deficient adenoviral vector of interest and that are generated using standard molecular biological techniques. The cell lines are further characterized in that they contain the complementing genes in a non-overlapping fashion, which minimizes, and practically eliminates, the possibility of the vector genome recombining with the cellular DNA. Accordingly, replication competent adenoviruses are not present in vector stocks, which are, therefore, suitable for certain therapeutic purposes, especially gene therapy purposes. This also avoids the replication of the adenoviruses in non-complementing cells.

[0036] The complementing cell line must be one that is capable of expressing the products of the deficient adenoviral gene functions at the appropriate level for those products in order to generate a high titer stock of recombinant adenoviral vector. For example, it is necessary to express the E2A product, DBP, at stoichiometric levels, i.e., relatively high levels, for adenoviral DNA replication, but the E2B product, Ad pol, is necessary at only catalytic levels, i.e., relatively low levels, for adenoviral DNA replication. Not only must the level of the product be appropriate, the temporal expression of the product must be consistent with that seen in normal viral infection of a cell to assure a high titer stock of recombinant adenoviral vector. For example, the components necessary for viral DNA replication must be expressed before those necessary for virion assembly. In order to avoid cellular toxicity, which often accompanies high levels of expression of the viral products, and to regulate the temporal expression of the products, inducible promoter systems are used. For example, the sheep metallothionine inducible promoter system can be used to express the complete E4 region, the open reading frame 6 of the E4 region, and the E2A region. Other examples of suitable inducible promoter systems include, but are not limited to, the bacterial lac operon, the tetracycline operon, the T7 polymerase system, and combinations and chimeric constructs of eukaryotic and prokaryotic transcription factors, repressors, and other components. Where the viral product to be expressed is highly toxic, it is desirable to use a bipartite inducible system, wherein the inducer is carried in a viral vector and the inducible product is carried within the chromatin of the complementing cell line. Repressible/inducible expression systems, such as the tetracycline expression system and lac expression system also can be used.

Methods of Use

[0037] The present invention provides a method of treating a tumor or cancer in a host comprising administering an anti-cancer or anti-tumor effective amount of the adenoviral vector of the present invention to a host in need thereof.

[0038] One skilled in the art will appreciate that suitable methods of administering a replication deficient adenoviral vector of the present invention to an animal for therapeutic or prophylactic purposes, e.g., gene therapy, vaccination, and the like (see, for example, Rosenfeld et al., Science, 252, 431-434 (1991), Jaffe et al., Clin. Res., 39(2), 302A (1991), Rosenfeld et al., Clin. Res., 39(2), 311A (1991), Berkner, BioTechniques, 6, 616-629 (1988)), are available, and, although more than one route can be used to administer the vector, a particular route can provide a more immediate and more effective reaction than another route.

[0039] The present invention provides a pharmaceutical composition comprising the adenoviral vector of the present invention and a carrier, especially a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). Pharmaceutically acceptable carriers are well-known to those who are skilled in the art and are readily available. The choice of carrier will be determined in part by the particular method used to administer the pharmaceutical composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations and methods are merely exemplary and are in no way limiting. However, oral, injectable and aerosol formulations are preferred.

[0040] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0041] The vectors of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

[0042] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0043] Additionally, the vectors employed in the present invention can be made into suppositories by mixing with a variety of bases such as emulsifying bases or watersoluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0044] The dose administered to an animal, particularly a human, in the context of the present invention will vary with the particular adenoviral vector, the composition containing the adenoviral vector, the method of administration, and the particular site and organism being treated. The dose should be sufficient to effect a desirable response, e.g., therapeutic or prophylactic response, within a desirable time frame.

[0045] The present method of treating a tumor or cancer in a host further can comprise the administration (i.e., pre-administration, co-administration, and/or post-administration) of other treatments and/or agents to modify (e.g., enhance) the effectiveness thereof. For example, an adenoviral vector of the present invention, particularly a vector comprising a nucleic acid sequence coding for TNF that is operably linked to a radiation inducible promoter, can be administered in conjunction with the administration of radiation. The radiation can be administered in any suitable manner, for example, by exposure to an external source of radiation (e.g., infrared radiation), or through the use of an internal source of radiation (e.g., through the chemical or surgical administration of a source of radiation). For instance, the adenoviral vector of the present invention can be used in conjunction with brachytherapy, wherein a radioactive source is placed (i.e., implanted) in or near a tumor to deliver a high, localized dose of radiation. Radiation is desirably administered in a dose sufficient to induce the production of a therapeutic level of TNF in the host.

[0046] The method of the present invention, additionally or alternatively to the administration of radiation, further can comprise the administration of other substances which locally or systemically alter (i.e., diminish or enhance) the effect of TNF in vivo. For example, substances that diminish the systemic effect of TNF can be used to control the level of systemic toxicity expressed in a host. Likewise, substances that enhance the local effect of TNF can be used to reduce the level of TNF required to produce a prophylactic or therapeutic effect in a host. Such substances include TNF antagonists, for example, soluble TNF receptors or anti-TNF antibodies, and TNF agonists. Other suitable antagonists, agonists, and other substances that alter the effect of TNF are available and generally known in the art.

[0047] The replication deficient adenoviral vectors of the present invention also have utility in vitro. For example, they can be used to study adenoviral gene function and assembly, the production of TNF, or the expression of other foreign nucleic acid sequences in a suitable target cell. One of ordinary skill can identify a suitable target cell by selecting one that can be transfected by the adenoviral vector, resulting in expression of the thereby inserted adenoviral nucleic acid sequence complement. Preferably, a suitable target cell is selected that has receptors for attachment and penetration of adenovirus into a cell. Such cells include, but are not limited to, those originally isolated from any mammal. Once the suitable target cell has been selected, the target cell is contacted with an adenoviral vector of the present invention, thereby effecting transfection or infection, respectively. Expression, toxicity, and other parameters relating to the insertion and activity of the nucleic acid sequence encoding TNF, or other foreign nucleic acid sequences, in the target cell then is measured using conventional methods well known in the art. For example, the adenoviral vector of the present invention can be used to study the interaction and targeting of TNF with respect to other cellular molecules. In so doing, researchers can learn and elucidate the phenomenology concerning adenoviral infection as well as the efficacy and effect of expression of various foreign nucleic acid sequences introduced by the adenoviral vector in various cell types that are explanted from various organisms and studied in tissue culture.

[0048] Moreover, cells explanted or removed from a patient having a disease that is suitably treated by gene therapy in the context of the present invention usefully are manipulated in vitro. For example, cells cultured in vitro from such an individual are placed in contact with an adenoviral vector of the present invention under suitable conditions to effect transfection, which are readily determined by one of ordinary skill in the art. Such contact suitably results in transfection of the vector into the cultured cells, where the transfected cells are selected using a suitable marker and selective culturing conditions. In so doing, using standard methods to test for vitality of the cells and thus measure toxicity and to test for presence of gene products of the foreign nucleic acid sequences of the vector of the present invention and thus measure expression, the cells of the individual are tested for compatibility with, expression in, and toxicity of the vector of the present invention, thereby providing information as to the appropriateness and efficacy of treatment of the individual with the vector system so tested. Such explanted and transfected cells, in addition to serving to test the potential efficacy/toxicity of a given gene therapy regime, also can be returned to an in vivo position within the body of the individual. Such cells so returned to the individual can be returned unaltered and unadorned except for the in vitro transfection thereof, or encased by or embedded in a matrix that keeps them separate from other tissues and cells of the individual's body. Such a matrix can be any suitable biocompatible material, including collagen, cellulose, and the like. Of course, alternatively or in addition, preferably after a positive response to the in vitro test, the transfection can be implemented in vivo by administration means as detailed hereinabove.

Further Aspects of the Present Invention

[0049] As those of ordinary skill in the art will appreciate, the adenoviral vector, and methods involving the same, provided by the present invention can comprise any combination or permutation of the elements described herein. A vector having a preferred configuration of such elements is schematically represented by FIG. 1B. Furthermore, a vector having such configuration of elements is provided by a vector that comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 1. However, many modifications and variations of the present illustrative nucleic acid sequence are possible. For example, the degeneracy of the genetic code allows for the substitution of nucleotides throughout polypeptide coding regions, as well as in the translational stop signal, without alteration of the encoded polypeptide coding sequence. Such substitutable sequences can be deduced from the known amino acid or nucleic acid sequence of a given gene and can be constructed by conventional synthetic or site-specific mutagenesis procedures. Synthetic DNA methods can be carried out in substantial accordance with the procedures of Itakura et al., Science, 198, 1056-1063 (1977), and Crea et al., Proc. Natl. Acad. Sci. USA, 75, 5765-5769 (1978). Site-specific mutagenesis procedures are described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (2d ed. 1989). Therefore, the present invention is in no way limited to the nucleic acid sequence specifically exemplified herein. Exemplified vectors are for gene therapy of tumors and/or cancer and, therefore, contain and express the TNF gene. However, the vectors described also can comprise genes used to treat other similar or different diseases and/or afflictions including, but not limited to, other chronic lung diseases, such as emphysema, asthma, adult respiratory distress syndrome, and chronic bronchitis, as well as coronary heart disease, and other afflictions suitably treated or prevented by gene therapy, vaccination, and the like. Accordingly, any gene or nucleic acid sequence can be inserted into the adenoviral TNF vector.

[0050] The adenoviral vector can be modified in other ways without departing from the scope and spirit of the present invention. For example, the coat protein of the present inventive adenoviral vector can be manipulated to alter the binding specificity or recognition of the virus for a viral receptor on a potential host cell. Such manipulations can include deletion of regions of the fiber, penton, or hexon, insertions of various native or non-native ligands into portions of the coat protein, and the like. Manipulation of the coat protein can broaden the range of cells infected by a viral vector or enable targeting of a viral vector to a specific cell type.

[0051] For example, the vector can comprise a chimeric coat protein (e.g., a fiber, hexon or penton protein), which differs from the wild-type (i.e., native) coat protein by the introduction of a nonnative amino acid sequence, preferably at or near the carboxyl terminus. Preferably, the nonnative amino acid sequence is inserted into or in place of an internal coat protein sequence. The resultant chimeric viral coat protein is able to direct entry into cells of the adenoviral vector comprising the coat protein that is more efficient than entry into cells of an adenoviral vector that is identical except for comprising a wild-type viral coat protein rather than the chimeric viral coat protein.

[0052] The chimeric virus coat protein desirably binds a novel endogenous binding site present on the cell surface. A result of this increased efficiency of entry is that the adenoviral virus can bind to and enter numerous cell types which a virus comprising wild-type coat protein typically cannot enter or can enter with only a low efficiency.

[0053] Alternatively, the adenoviral vector of the present invention can comprise a chimeric virus coat protein that is not selective for a specific type of eukaryotic cell. Such chimeric coat protein differs from the wild-type coat protein by an insertion of a normative amino acid sequence into or in place of an internal coat protein sequence. In a vector comprising a non-selective chimeric coat protein, the virus coat efficiently binds to a broader range of eukaryotic cells than a wild-type virus coat, such as described in International Patent Application WO 97/20051.

[0054] Specificity of binding of an adenovirus to a given cell also can be adjusted by use of an adenovirus comprising a short-shafted adenoviral fiber gene, as discussed in U.S. Pat. No. 5,962,311. Use of an adenovirus comprising a short-shafted adenoviral fiber gene reduces the level or efficiency of adenoviral fiber binding to its cell-surface receptor and increases adenoviral penton base binding to its cell-surface receptor, thereby increasing the specificity of binding of the adenovirus to a given cell. Alternatively, use of an adenovirus comprising a short-shafted fiber enables targeting of the adenovirus to a desired cell-surface receptor by the introduction of a normative amino acid sequence either into the penton base or the fiber knob.

[0055] In addition, the ability of a viral vector to recognize a potential host cell can be modulated without genetic manipulation of the coat protein. For instance, complexing an adenovirus with a bispecific molecule comprising a penton base-binding domain and a domain that selectively binds a particular cell surface binding site enables one of ordinary skill in the art to target the vector to a particular cell type.

[0056] Many modifications to a viral vector, specifically an adenoviral vector, are known in the art. Suitable modifications for an adenoviral vector include those modifications described in U.S. Pat. Nos. 5,559,099; 5,731,190; 5,712,136; 5,770,442; 5,846,782; 5,926,311; and 5,965,541 and International Patent Applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, and WO 98/54346.

[0057] The following examples further illustrate the present invention and, of course, should not be construed as in any way limiting its scope. Enzymes referred to in the examples are available, unless otherwise indicated, from Bethesda Research Laboratories (BRL), Gaithersburg, Md., New England Biolabs Inc. (NEB), Beverly, Mass., or Boehringer Mannheim Biochemicals (BMB), Indianapolis, Ind., and are used in substantial accordance with the manufacturer's recommendations. Many of the techniques employed herein are well known to those in the art. Molecular biology techniques are described in detail in suitable laboratory manuals, such as Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (2d ed. 1989), and Current Protocols in Molecular Biology (Ausubel et al., eds. (1987)).

EXAMPLE 1

[0058] The following example demonstrates the preparation of an adenoviral vector in accordance with the present invention. A first plasmid (base plasmid) comprising a type-5 adenoviral genome was prepared having deficiencies in the E1, E3, and E4 regions, wherein the E3 region was entirely deleted and the E4 region comprised an SV40 polyadenylation sequence. A second plasmid (donor plasmid) was prepared comprising an expression cassette having an Egr-1 radiation-inducible promoter operably linked to a nucleic acid sequence encoding TNF-α. The donor plasmid was digested with Drd-I restriction endonuclease, and a nucleic acid segment of approximately 6.5 kbp comprising the Egr-I/TNF-α expression cassette was isolated by gel electrophoresis. The base plasmid was digested using Swa-I restriction endonuclease, and the linearized plasmid was de-phosphorylated. Competent E. coli BJIq cells were transformed with 50 ng of the 6.5 kbp (approx.) donor plasmid fragment and 50 ng of the linearized base plasmid. The transformed cells were plated on luria/SOC broth plates containing 50 μg/μl of kanamycin and 50 μg/μl tetracycline. The resulting colonies were screened for recombinants, and the positive recombinants were grown on luria broth/kanamycin plates.

[0059] The recombinant plasmid DNA was isolated from the transformed E. coli BJIq cells by standard techniques. Using standard procedures, the isolated plasmid DNA was linearized by digestion with Pac-I restriction endonuclease, and the linearized plasmid DNA was transfected in 293-ORF6 cells (derived from type 293 human embryonic kidney cells), which compliment for E1 and E4 deficiencies, to produce an adenoviral Egr-1/TNF-α E1⁻, E3⁻, E4⁻ vector having the sequence of SEQ ID NO: 1.

EXAMPLE 2

[0060] The following example demonstrates the use of an adenoviral vector prepared in accordance with the present invention to treat or prevent tumors in a host.

[0061] Three treatment groups were established, each comprising eight nude mice having radio-resistant human squameous tumor cell line (SQ-20B) xenograft tumors. The first treatment group received a dose of 5×10¹⁰ particle units (PU) of the adenoviral particles of Example 1 (in a total volume of 32 μl with a viral buffer) by injection directly into the tumor tissue at five sites (four injections around the periphery of each tumor and one injection into the center of each tumor) at days 0, 4, 7, and 11. The second treatment group received the same doses of adenoviral vector administered in conjunction with exposure of the tumor to 5 Gy of infrared radiation on days 0-4 and 7-9 (totaling 40 Gy of radiation). The third treatment group received only the infrared radiation.

[0062] Significant tumor regression was observed in the first treatment group by day four, while no regression was observed in the second treatment group until day 11. At day 11, one animal in the first treatment group and three animals in the second treatment group had no visible tumor. After 62 days, all eight mice in the second treatment group were cured (i.e., no visible tumors were present), while six mice of the first group were cured, and only one mouse of the third group (no vector) was cured.

[0063] The results of this example demonstrate that an adenoviral vector according to the present invention can be successfully used to treat tumors in a host.

EXAMPLE 3

[0064] The following example demonstrates the use of an adenoviral vector prepared in accordance with the present invention to treat or prevent tumors in a host.

[0065] Radio-resistant human esophageal tumor cells derived from patient samples were injected into the right hind limb of nude mice in a single dose of 5×10⁶ cells, and the mice were randomly divided into four treatment groups. Group 1 was treated only with a buffer solution, which was administered by direct injection into the tumor on day 0 and day 3. Group 2 received infrared radiation treatment consisting of 4 Gy doses of radiation administered on days 0, 1, 3 and 4 (for a total IR dose of 16 Gy). Group 3 was treated with the adenoviral vector of Example 1 (at 4×10⁸ PU), which was administered by direct injection into the tumor on day 0 and day 3. Group 4 received the adenoviral vector treatment and the infrared radiation treatment.

[0066] Tumor volumes and animals weights were measured over 48 days. The tumor volumes were plotted against time to yield the tumor volume curves presented in FIG. 2, wherein the diamond-shaped data points (υ) represent data from group 1, the circular data points (λ) represent data from group 2, triangular data points (σ) represent data from group 3, and the square data points (ν) represent data from group 4. All animals remained healthy and no untoward effects were observed throughout the study. Animals from groups 1 and 2, however, were sacrificed at day 36 due to the large tumor burden (greater than 10% of body weight).

[0067] The tumor volume curves demonstrate substantially less tumor growth in animals treated with the vector and radiation (group 4) as compared to those treated with radiation alone (group 2) or the vector alone (group 3). This data demonstrates that the adenoviral vector of the present invention can be successfully used to treat a tumor in a host.

EXAMPLE 4

[0068] The following example demonstrates the use of an adenoviral vector prepared in accordance with the present invention to treat or prevent tumors in a host.

[0069] Radio-resistant esophageal tumor cells derived from patient samples were injected into the right hind limb of nude mice in a single dose of 5×10⁶ cells, and the mice were randomly divided into four treatment groups. Group 1 was treated only with a buffer solution, which was administered by direct injection into the tumor on days 0, 3, 7 and 10. Group 2 received infrared radiation treatment consisting of 3 Gy doses of radiation administered on days 0, 1, 3, 4, 7, 8, 10 and 11 (for a total IR dose of 24 Gy). Group 3 was treated with the adenoviral vector of Example 1 (at 4×10⁸ PU), which was administered by direct injection into the tumor on days 0, 3, 7 and 10. Group 4 received the adenoviral vector treatment and the infrared radiation treatment.

[0070] Tumor volumes and animals weights were measured over 22 days. The tumor volumes were plotted against time to provide the tumor volume curves presented in FIG. 3, wherein the diamond-shaped data points (υ) represent data from group 1, the circular data points (λ) represent data from group 2, triangular data points (σ) represent data from group 3, and the square data points (ν) represent data from group 4. All animals remained healthy and no untoward effects were observed. Animals from groups 1 and 2, however, were sacrificed at day 15 due to the large tumor burden (greater than 10% of body weight).

[0071] The tumor volume curves demonstrate substantially less tumor growth in animals treated with the vector and radiation (group 4) as compared to those treated with radiation alone (group 2) or the vector alone (group 3). This data demonstrates that the adenoviral vector of the present invention can be successfully used to treat a tumor in a host.

[0072] All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

[0073] While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

1 2 1 32798 DNA Artificial Sequence Synthetic construct 1 catcatcata atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt 60 tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg 120 atgttgcaag tgtggcggaa cacatgtaag cgccggatgt ggtaaaagtg acgtttttgg 180 tgtgcgccgg tgtatacggg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240 aaatttgggc gtaaccaagt aatatttggc cattttcgcg ggaaaactga ataagaggaa 300 gtgaaatctg aataattctg tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360 actttgaccg tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420 gggtcaaagt tggcgtttta ttattatagt cagctctaga ctagatgcgc cgacccggaa 480 acgccatata aggagcagga aggatccccc gccggaacag accttatttg ggcagcgcct 540 tatatggagt ggcccaatat ggccctgccg cttccggctc tgggaggagg ggcgagcggg 600 ggttggggcg ggggcaagct gggaactcca ggcgcctggc ccgggaggcc actgctgctg 660 ttccaatact aggctttcca ggagcctgag cgctcgcgat gccggagcgg gtcgcagggt 720 ggaggtgccc accactcttg gatgggaggg cttcacgtca ctccgggtcc tcccggccgg 780 tccttccata ttagggcttc ctgcttccca tatatggcca tgtacgtcac ggcggaggcg 840 ggcccgtgct gttccagacc cttgaaatag aggccgattc ggggagtcgc gagagatccc 900 agcgcgcaga acttggggag ccgccgccgc gattcgccgc cgccgccagc ttccggtcga 960 ggaactgaaa aaccagaaag ttaactgggt aagtttagtc tttttgtctt ttatttcagg 1020 tcccggatcc ggtggtggtg caaatcaaag aactgctcct cagtggatgt tgcctttact 1080 tctaggcctg tacggaagtg ttacttctgc tctaaaagct gcggaattgt acccgcggcc 1140 gcaaagggaa caaaagctgg gtaccgagct cgaatggggg gggggggggg gtactgaccc 1200 acggctccac cctctctccc ctggaaagga caccatgagc actgaaagca tgatccggga 1260 cgtggagctg gccgaggagg cgctccccaa gaagacaggg gggccccagg gctccaggcg 1320 gtgcttgttc ctcagcctct tctccttcct gatcgtggca ggcgccacca cgctcttctg 1380 cctgctgcac tttggagtga tcggccccca gagggaagag tcccccaggg acctctctct 1440 aatcagccct ctggcccagg cagtcagatc atcttctcga accccgagtg acaagcctgt 1500 agcccatgtt gtagcaaacc ctcaagctga ggggcagctc cagtggctga accgccgggc 1560 caatgccctc ctggccaatg gcgtggagct gagagataac cagctggtgg tgccatcaga 1620 gggcctgtac ctcatctact cccaggtcct cttcaagggc caaggctgcc cctccaccca 1680 tgtgctcctc acccacacca tcagccgcat cgccgtctcc taccagacca aggtcaacct 1740 cctctctgcc atcaagagcc cctgccagag ggagacccca gagggggctg aggccaagcc 1800 ctggtatgag cccatctatc tgggaggggt cttccagctg gagaagggtg accgactcag 1860 cgctgagatc aatcggcccg actatctcga ctttgccgag tctgggcagg tctactttgg 1920 gatcattgcc ctgtgaggag gacgaacatc caaccttccc aaacgcctcc cctgccccaa 1980 tccctttatt accccctcct tcagacaccc tcaacctctt ctggctcaaa aagagaattg 2040 ggggcttagg gtcggaaccc aagcttgata tcgaattcct gcagcccggg ggatccacta 2100 gttctagagc ggccgcgggg atccagacat gataagatac attgatgagt ttggacaaac 2160 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 2220 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 2280 gtttcaggtt cagggggagg tgtgggaggt tttttcggat cctctagagt cgactagagt 2340 ggaaggtgct gaggtacgat gagacccgca ccaggtgcag accctgcgag tgtggcggta 2400 aacatattag gaaccagcct gtgatgctgg atgtgaccga ggagctgagg cccgatcact 2460 tggtgctggc ctgcacccgc gctgagtttg gctctagcga tgaagataca gattgaggta 2520 ctgaaatgtg tgggcgtggc ttaagggtgg gaaagaatat ataaggtggg ggtcttatgt 2580 agttttgtat ctgttttgca gcagccgccg ccgccatgag caccaactcg tttgatggaa 2640 gcattgtgag ctcatatttg acaacgcgca tgcccccatg ggccggggtg cgtcagaatg 2700 tgatgggctc cagcattgat ggtcgccccg tcctgcccgc aaactctact accttgacct 2760 acgagaccgt gtctggaacg ccgttggaga ctgcagcctc cgccgccgct tcagccgctg 2820 cagccaccgc ccgcgggatt gtgactgact ttgctttcct gagcccgctt gcaagcagtg 2880 cagcttcccg ttcatccgcc cgcgatgaca agttgacggc tcttttggca caattggatt 2940 ctttgacccg ggaacttaat gtcgtttctc agcagctgtt ggatctgcgc cagcaggttt 3000 ctgccctgaa ggcttcctcc cctcccaatg cggtttaaaa cataaataaa aaaccagact 3060 ctgtttggat ttggatcaag caagtgtctt gctgtcttta tttaggggtt ttgcgcgcgc 3120 ggtaggcccg ggaccagcgg tctcggtcgt tgagggtcct gtgtattttt tccaggacgt 3180 ggtaaaggtg actctggatg ttcagataca tgggcataag cccgtctctg gggtggaggt 3240 agcaccactg cagagcttca tgctgcgggg tggtgttgta gatgatccag tcgtagcagg 3300 agcgctgggc gtggtgccta aaaatgtctt tcagtagcaa gctgattgcc aggggcaggc 3360 ccttggtgta agtgtttaca aagcggttaa gctgggatgg gtgcatacgt ggggatatga 3420 gatgcatctt ggactgtatt tttaggttgg ctatgttccc agccatatcc ctccggggat 3480 tcatgttgtg cagaaccacc agcacagtgt atccggtgca cttgggaaat ttgtcatgta 3540 gcttagaagg aaatgcgtgg aagaacttgg agacgccctt gtgacctcca agattttcca 3600 tgcattcgtc cataatgatg gcaatgggcc cacgggcggc ggcctgggcg aagatatttc 3660 tgggatcact aacgtcatag ttgtgttcca ggatgagatc gtcataggcc atttttacaa 3720 agcgcgggcg gagggtgcca gactgcggta taatggttcc atccggccca ggggcgtagt 3780 taccctcaca gatttgcatt tcccacgctt tgagttcaga tggggggatc atgtctacct 3840 gcggggcgat gaagaaaacg gtttccgggg taggggagat cagctgggaa gaaagcaggt 3900 tcctgagcag ctgcgactta ccgcagccgg tgggcccgta aatcacacct attaccgggt 3960 gcaactggta gttaagagag ctgcagctgc cgtcatccct gagcaggggg gccacttcgt 4020 taagcatgtc cctgactcgc atgttttccc tgaccaaatc cgccagaagg cgctcgccgc 4080 ccagcgatag cagttcttgc aaggaagcaa agtttttcaa cggtttgaga ccgtccgccg 4140 taggcatgct tttgagcgtt tgaccaagca gttccaggcg gtcccacagc tcggtcacct 4200 gctctacggc atctcgatcc agcatatctc ctcgtttcgc gggttggggc ggctttcgct 4260 gtacggcagt agtcggtgct cgtccagacg ggccagggtc atgtctttcc acgggcgcag 4320 ggtcctcgtc agcgtagtct gggtcacggt gaaggggtgc gctccgggct gcgcgctggc 4380 cagggtgcgc ttgaggctgg tcctgctggt gctgaagcgc tgccggtctt cgccctgcgc 4440 gtcggccagg tagcatttga ccatggtgtc atagtccagc ccctccgcgg cgtggccctt 4500 ggcgcgcagc ttgcccttgg aggaggcgcc gcacgagggg cagtgcagac ttttgagggc 4560 gtagagcttg ggcgcgagaa ataccgattc cggggagtag gcatccgcgc cgcaggcccc 4620 gcagacggtc tcgcattcca cgagccaggt gagctctggc cgttcggggt caaaaaccag 4680 gtttccccca tgctttttga tgcgtttctt acctctggtt tccatgagcc ggtgtccacg 4740 ctcggtgacg aaaaggctgt ccgtgtcccc gtatacagac ttgagaggcc tgtcctcgag 4800 cggtgttccg cggtcctcct cgtatagaaa ctcggaccac tctgagacaa aggctcgcgt 4860 ccaggccagc acgaaggagg ctaagtggga ggggtagcgg tcgttgtcca ctagggggtc 4920 cactcgctcc agggtgtgaa gacacatgtc gccctcttcg gcatcaagga aggtgattgg 4980 tttgtaggtg taggccacgt gaccgggtgt tcctgaaggg gggctataaa agggggtggg 5040 ggcgcgttcg tcctcactct cttccgcatc gctgtctgcg agggccagct gttggggtga 5100 gtactccctc tgaaaagcgg gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga 5160 ggaggatttg atattcacct ggcccgcggt gatgcctttg agggtggccg catccatctg 5220 gtcagaaaag acaatctttt tgttgtcaag cttggtggca aacgacccgt agagggcgtt 5280 ggacagcaac ttggcgatgg agcgcagggt ttggtttttg tcgcgatcgg cgcgctcctt 5340 ggccgcgatg tttagctgca cgtattcgcg cgcaacgcac cgccattcgg gaaagacggt 5400 ggtgcgctcg tcgggcacca ggtgcacgcg ccaaccgcgg ttgtgcaggg tgacaaggtc 5460 aacgctggtg gctacctctc cgcgtaggcg ctcgttggtc cagcagaggc ggccgccctt 5520 gcgcgagcag aatggcggta gggggtctag ctgcgtctcg tccggggggt ctgcgtccac 5580 ggtaaagacc ccgggcagca ggcgcgcgtc gaagtagtct atcttgcatc cttgcaagtc 5640 tagcgcctgc tgccatgcgc gggcggcaag cgcgcgctcg tatgggttga gtgggggacc 5700 ccatggcatg gggtgggtga gcgcggaggc gtacatgccg caaatgtcgt aaacgtagag 5760 gggctctctg agtattccaa gatatgtagg gtagcatctt ccaccgcgga tgctggcgcg 5820 cacgtaatcg tatagttcgt gcgagggagc gaggaggtcg ggaccgaggt tgctacgggc 5880 gggctgctct gctcggaaga ctatctgcct gaagatggca tgtgagttgg atgatatggt 5940 tggacgctgg aagacgttga agctggcgtc tgtgagacct accgcgtcac gcacgaagga 6000 ggcgtaggag tcgcgcagct tgttgaccag ctcggcggtg acctgcacgt ctagggcgca 6060 gtagtccagg gtttccttga tgatgtcata cttatcctgt cccttttttt tccacagctc 6120 gcggttgagg acaaactctt cgcggtcttt ccagtactct tggatcggaa acccgtcggc 6180 ctccgaacgg taagagccta gcatgtagaa ctggttgacg gcctggtagg cgcagcatcc 6240 cttttctacg ggtagcgcgt atgcctgcgc ggccttccgg agcgaggtgt gggtgagcgc 6300 aaaggtgtcc ctgaccatga ctttgaggta ctggtatttg aagtcagtgt cgtcgcatcc 6360 gccctgctcc cagagcaaaa agtccgtgcg ctttttggaa cgcggatttg gcagggcgaa 6420 ggtgacatcg ttgaagagta tctttcccgc gcgaggcata aagttgcgtg tgatgcggaa 6480 gggtcccggc acctcggaac ggttgttaat tacctgggcg gcgagcacga tctcgtcaaa 6540 gccgttgatg ttgtggccca caatgtaaag ttccaagaag cgcgggatgc ccttgatgga 6600 aggcaatttt ttaagttcct cgtaggtgag ctcttcaggg gagctgagcc cgtgctctga 6660 aagggcccag tctgcaagat gagggttgga agcgacgaat gagctccaca ggtcacgggc 6720 cattagcatt tgcaggtggt cgcgaaaggt cctaaactgg cgacctatgg ccattttttc 6780 tggggtgatg cagtagaagg taagcgggtc ttgttcccag cggtcccatc caaggttcgc 6840 ggctaggtct cgcgcggcag tcactagagg ctcatctccg ccgaacttca tgaccagcat 6900 gaagggcacg agctgcttcc caaaggcccc catccaagta taggtctcta catcgtaggt 6960 gacaaagaga cgctcggtgc gaggatgcga gccgatcggg aagaactgga tctcccgcca 7020 ccaattggag gagtggctat tgatgtggtg aaagtagaag tccctgcgac gggccgaaca 7080 ctcgtgctgg cttttgtaaa aacgtgcgca gtactggcag cggtgcacgg gctgtacatc 7140 ctgcacgagg ttgacctgac gaccgcgcac aaggaagcag agtgggaatt tgagcccctc 7200 gcctggcggg tttggctggt ggtcttctac ttcggctgct tgtccttgac cgtctggctg 7260 ctcgagggga gttacggtgg atcggaccac cacgccgcgc gagcccaaag tccagatgtc 7320 cgcgcgcggc ggtcggagct tgatgacaac atcgcgcaga tgggagctgt ccatggtctg 7380 gagctcccgc ggcgtcaggt caggcgggag ctcctgcagg tttacctcgc atagacgggt 7440 cagggcgcgg gctagatcca ggtgatacct aatttccagg ggctggttgg tggcggcgtc 7500 gatggcttgc aagaggccgc atccccgcgg cgcgactacg gtaccgcgcg gcgggcggtg 7560 ggccgcgggg gtgtccttgg atgatgcatc taaaagcggt gacgcgggcg agcccccgga 7620 ggtagggggg gctccggacc cgccgggaga gggggcaggg gcacgtcggc gccgcgcgcg 7680 ggcaggagct ggtgctgcgc gcgtaggttg ctggcgaacg cgacgacgcg gcggttgatc 7740 tcctgaatct ggcgcctctg cgtgaagacg acgggcccgg tgagcttgag cctgaaagag 7800 agttcgacag aatcaatttc ggtgtcgttg acggcggcct ggcgcaaaat ctcctgcacg 7860 tctcctgagt tgtcttgata ggcgatctcg gccatgaact gctcgatctc ttcctcctgg 7920 agatctccgc gtccggctcg ctccacggtg gcggcgaggt cgttggaaat gcgggccatg 7980 agctgcgaga aggcgttgag gcctccctcg ttccagacgc ggctgtagac cacgccccct 8040 tcggcatcgc gggcgcgcat gaccacctgc gcgagattga gctccacgtg ccgggcgaag 8100 acggcgtagt ttcgcaggcg ctgaaagagg tagttgaggg tggtggcggt gtgttctgcc 8160 acgaagaagt acataaccca gcgtcgcaac gtggattcgt tgatatcccc caaggcctca 8220 aggcgctcca tggcctcgta gaagtccacg gcgaagttga aaaactggga gttgcgcgcc 8280 gacacggtta actcctcctc cagaagacgg atgagctcgg cgacagtgtc gcgcacctcg 8340 cgctcaaagg ctacaggggc ctcttcttct tcttcaatct cctcttccat aagggcctcc 8400 ccttcttctt cttctggcgg cggtggggga ggggggacac ggcggcgacg acggcgcacc 8460 gggaggcggt cgacaaagcg ctcgatcatc tccccgcggc gacggcgcat ggtctcggtg 8520 acggcgcggc cgttctcgcg ggggcgcagt tggaagacgc cgcccgtcat gtcccggtta 8580 tgggttggcg gggggctgcc atgcggcagg gatacggcgc taacgatgca tctcaacaat 8640 tgttgtgtag gtactccgcc gccgagggac ctgagcgagt ccgcatcgac cggatcggaa 8700 aacctctcga gaaaggcgtc taaccagtca cagtcgcaag gtaggctgag caccgtggcg 8760 ggcggcagcg ggcggcggtc ggggttgttt ctggcggagg tgctgctgat gatgtaatta 8820 aagtaggcgg tcttgagacg gcggatggtc gacagaagca ccatgtcctt gggtccggcc 8880 tgctgaatgc gcaggcggtc ggccatgccc caggcttcgt tttgacatcg gcgcaggtct 8940 ttgtagtagt cttgcatgag cctttctacc ggcacttctt cttctccttc ctcttgtcct 9000 gcatctcttg catctatcgc tgcggcggcg gcggagtttg gccgtaggtg gcgccctctt 9060 cctcccatgc gtgtgacccc gaagcccctc atcggctgaa gcagggctag gtcggcgaca 9120 acgcgctcgg ctaatatggc ctgctgcacc tgcgtgaggg tagactggaa gtcatccatg 9180 tccacaaagc ggtggtatgc gcccgtgttg atggtgtaag tgcagttggc cataacggac 9240 cagttaacgg tctggtgacc cggctgcgag agctcggtgt acctgagacg cgagtaagcc 9300 ctcgagtcaa atacgtagtc gttgcaagtc cgcaccaggt actggtatcc caccaaaaag 9360 tgcggcggcg gctggcggta gaggggccag cgtagggtgg ccggggctcc gggggcgaga 9420 tcttccaaca taaggcgatg atatccgtag atgtacctgg acatccaggt gatgccggcg 9480 gcggtggtgg aggcgcgcgg aaagtcgcgg acgcggttcc agatgttgcg cagcggcaaa 9540 aagtgctcca tggtcgggac gctctggccg gtcaggcgcg cgcaatcgtt gacgctctag 9600 ccgtgcaaaa ggagagcctg taagcgggca ctcttccgtg gtctggtgga taaattcgca 9660 agggtatcat ggcggacgac cggggttcga gccccgtatc cggccgtccg ccgtgatcca 9720 tgcggttacc gcccgcgtgt cgaacccagg tgtgcgacgt cagacaacgg gggagtgctc 9780 cttttggctt ccttccaggc gcggcggctg ctgcgctagc ttttttggcc actggccgcg 9840 cgcagcgtaa gcggttaggc tggaaagcga aagcattaag tggctcgctc cctgtagccg 9900 gagggttatt ttccaagggt tgagtcgcgg gacccccggt tcgagtctcg gaccggccgg 9960 actgcggcga acgggggttt gcctccccgt catgcaagac cccgcttgca aattcctccg 10020 gaaacaggga cgagcccctt ttttgctttt cccagatgca tccggtgctg cggcagatgc 10080 gcccccctcc tcagcagcgg caagagcaag agcagcggca gacatgcagg gcaccctccc 10140 ctcctcctac cgcgtcagga ggggcgacat ccgcggttga cgcggcagca gatggtgatt 10200 acgaaccccc gcggcgccgg gcccggcact acctggactt ggaggagggc gagggcctgg 10260 cgcggctagg agcgccctct cctgagcggt acccaagggt gcagctgaag cgtgatacgc 10320 gtgaggcgta cgtgccgcgg cagaacctgt ttcgcgaccg cgagggagag gagcccgagg 10380 agatgcggga tcgaaagttc cacgcagggc gcgagctgcg gcatggcctg aatcgcgagc 10440 ggttgctgcg cgaggaggac tttgagcccg acgcgcgaac cgggattagt cccgcgcgcg 10500 cacacgtggc ggccgccgac ctggtaaccg catacgagca gacggtgaac caggagatta 10560 actttcaaaa aagctttaac aaccacgtgc gtacgcttgt ggcgcgcgag gaggtggcta 10620 taggactgat gcatctgtgg gactttgtaa gcgcgctgga gcaaaaccca aatagcaagc 10680 cgctcatggc gcagctgttc cttatagtgc agcacagcag ggacaacgag gcattcaggg 10740 atgcgctgct aaacatagta gagcccgagg gccgctggct gctcgatttg ataaacatcc 10800 tgcagagcat agtggtgcag gagcgcagct tgagcctggc tgacaaggtg gccgccatca 10860 actattccat gcttagcctg ggcaagtttt acgcccgcaa gatataccat accccttacg 10920 ttcccataga caaggaggta aagatcgagg ggttctacat gcgcatggcg ctgaaggtgc 10980 ttaccttgag cgacgacctg ggcgtttatc gcaacgagcg catccacaag gccgtgagcg 11040 tgagccggcg gcgcgagctc agcgaccgcg agctgatgca cagcctgcaa agggccctgg 11100 ctggcacggg cagcggcgat agagaggccg agtcctactt tgacgcgggc gctgacctgc 11160 gctgggcccc aagccgacgc gccctggagg cagctggggc cggacctggg ctggcggtgg 11220 cacccgcgcg cgctggcaac gtcggcggcg tggaggaata tgacgaggac gatgagtacg 11280 agccagagga cggcgagtac taagcggtga tgtttctgat cagatgatgc aagacgcaac 11340 ggacccggcg gtgcgggcgg cgctgcagag ccagccgtcc ggccttaact ccacggacga 11400 ctggcgccag gtcatggacc gcatcatgtc gctgactgcg cgcaatcctg acgcgttccg 11460 gcagcagccg caggccaacc ggctctccgc aattctggaa gcggtggtcc cggcgcgcgc 11520 aaaccccacg cacgagaagg tgctggcgat cgtaaacgcg ctggccgaaa acagggccat 11580 ccggcccgac gaggccggcc tggtctacga cgcgctgctt cagcgcgtgg ctcgttacaa 11640 cagcggcaac gtgcagacca acctggaccg gctggtgggg gatgtgcgcg aggccgtggc 11700 gcagcgtgag cgcgcgcagc agcagggcaa cctgggctcc atggttgcac taaacgcctt 11760 cctgagtaca cagcccgcca acgtgccgcg gggacaggag gactacacca actttgtgag 11820 cgcactgcgg ctaatggtga ctgagacacc gcaaagtgag gtgtaccagt ctgggccaga 11880 ctattttttc cagaccagta gacaaggcct gcagaccgta aacctgagcc aggctttcaa 11940 aaacttgcag gggctgtggg gggtgcgggc tcccacaggc gaccgcgcga ccgtgtctag 12000 cttgctgacg cccaactcgc gcctgttgct gctgctaata gcgcccttca cggacagtgg 12060 cagcgtgtcc cgggacacat acctaggtca cttgctgaca ctgtaccgcg aggccatagg 12120 tcaggcgcat gtggacgagc atactttcca ggagattaca agtgtcagcc gcgcgctggg 12180 gcaggaggac acgggcagcc tggaggcaac cctaaactac ctgctgacca accggcggca 12240 gaagatcccc tcgttgcaca gtttaaacag cgaggaggag cgcattttgc gctacgtgca 12300 gcagagcgtg agccttaacc tgatgcgcga cggggtaacg cccagcgtgg cgctggacat 12360 gaccgcgcgc aacatggaac cgggcatgta tgcctcaaac cggccgttta tcaaccgcct 12420 aatggactac ttgcatcgcg cggccgccgt gaaccccgag tatttcacca atgccatctt 12480 gaacccgcac tggctaccgc cccctggttt ctacaccggg ggattcgagg tgcccgaggg 12540 taacgatgga ttcctctggg acgacataga cgacagcgtg ttttccccgc aaccgcagac 12600 cctgctagag ttgcaacagc gcgagcaggc agaggcggcg ctgcgaaagg aaagcttccg 12660 caggccaagc agcttgtccg atctaggcgc tgcggccccg cggtcagatg ctagtagccc 12720 atttccaagc ttgatagggt ctcttaccag cactcgcacc acccgcccgc gcctgctggg 12780 cgaggaggag tacctaaaca actcgctgct gcagccgcag cgcgaaaaaa acctgcctcc 12840 ggcatttccc aacaacggga tagagagcct agtggacaag atgagtagat ggaagacgta 12900 cgcgcaggag cacagggacg tgccaggccc gcgcccgccc acccgtcgtc aaaggcacga 12960 ccgtcagcgg ggtctggtgt gggaggacga tgactcggca gacgacagca gcgtcctgga 13020 tttgggaggg agtggcaacc cgtttgcgca ccttcgcccc aggctgggga gaatgtttta 13080 aaaaaaaaaa agcatgatgc aaaataaaaa actcaccaag gccatggcac cgagcgttgg 13140 ttttcttgta ttccccttag tatgcggcgc gcggcgatgt atgaggaagg tcctcctccc 13200 tcctacgaga gtgtggtgag cgcggcgcca gtggcggcgg cgctgggttc tcccttcgat 13260 gctcccctgg acccgccgtt tgtgcctccg cggtacctgc ggcctaccgg ggggagaaac 13320 agcatccgtt actctgagtt ggcaccccta ttcgacacca cccgtgtgta cctggtggac 13380 aacaagtcaa cggatgtggc atccctgaac taccagaacg accacagcaa ctttctgacc 13440 acggtcattc aaaacaatga ctacagcccg ggggaggcaa gcacacagac catcaatctt 13500 gacgaccggt cgcactgggg cggcgacctg aaaaccatcc tgcataccaa catgccaaat 13560 gtgaacgagt tcatgtttac caataagttt aaggcgcggg tgatggtgtc gcgcttgcct 13620 actaaggaca atcaggtgga gctgaaatac gagtgggtgg agttcacgct gcccgagggc 13680 aactactccg agaccatgac catagacctt atgaacaacg cgatcgtgga gcactacttg 13740 aaagtgggca gacagaacgg ggttctggaa agcgacatcg gggtaaagtt tgacacccgc 13800 aacttcagac tggggtttga ccccgtcact ggtcttgtca tgcctggggt atatacaaac 13860 gaagccttcc atccagacat cattttgctg ccaggatgcg gggtggactt cacccacagc 13920 cgcctgagca acttgttggg catccgcaag cggcaaccct tccaggaggg ctttaggatc 13980 acctacgatg atctggaggg tggtaacatt cccgcactgt tggatgtgga cgcctaccag 14040 gcgagcttga aagatgacac cgaacagggc gggggtggcg caggcggcag caacagcagt 14100 ggcagcggcg cggaagagaa ctccaacgcg gcagccgcgg caatgcagcc ggtggaggac 14160 atgaacgatc atgccattcg cggcgacacc tttgccacac gggctgagga gaagcgcgct 14220 gaggccgaag cagcggccga agctgccgcc cccgctgcgc aacccgaggt cgagaagcct 14280 cagaagaaac cggtgatcaa acccctgaca gaggacagca agaaacgcag ttacaaccta 14340 ataagcaatg acagcacctt cacccagtac cgcagctggt accttgcata caactacggc 14400 gaccctcaga ccggaatccg ctcatggacc ctgctttgca ctcctgacgt aacctgcggc 14460 tcggagcagg tctactggtc gttgccagac atgatgcaag accccgtgac cttccgctcc 14520 acgcgccaga tcagcaactt tccggtggtg ggcgccgagc tgttgcccgt gcactccaag 14580 agcttctaca acgaccaggc cgtctactcc caactcatcc gccagtttac ctctctgacc 14640 cacgtgttca atcgctttcc cgagaaccag attttggcgc gcccgccagc ccccaccatc 14700 accaccgtca gtgaaaacgt tcctgctctc acagatcacg ggacgctacc gctgcgcaac 14760 agcatcggag gagtccagcg agtgaccatt actgacgcca gacgccgcac ctgcccctac 14820 gtttacaagg ccctgggcat agtctcgccg cgcgtcctat cgagccgcac tttttgagca 14880 agcatgtcca tccttatatc gcccagcaat aacacaggct ggggcctgcg cttcccaagc 14940 aagatgtttg gcggggccaa gaagcgctcc gaccaacacc cagtgcgcgt gcgcgggcac 15000 taccgcgcgc cctggggcgc gcacaaacgc ggccgcactg ggcgcaccac cgtcgatgac 15060 gccatcgacg cggtggtgga ggaggcgcgc aactacacgc ccacgccgcc accagtgtcc 15120 acagtggacg cggccattca gaccgtggtg cgcggagccc ggcgctatgc taaaatgaag 15180 agacggcgga ggcgcgtagc acgtcgccac cgccgccgac ccggcactgc cgcccaacgc 15240 gcggcggcgg ccctgcttaa ccgcgcacgt cgcaccggcc gacgggcggc catgcgggcc 15300 gctcgaaggc tggccgcggg tattgtcact gtgcccccca ggtccaggcg acgagcggcc 15360 gccgcagcag ccgcggccat tagtgctatg actcagggtc gcaggggcaa cgtgtattgg 15420 gtgcgcgact cggttagcgg cctgcgcgtg cccgtgcgca cccgcccccc gcgcaactag 15480 attgcaagaa aaaactactt agactcgtac tgttgtatgt atccagcggc ggcggcgcgc 15540 aacgaagcta tgtccaagcg caaaatcaaa gaagagatgc tccaggtcat cgcgccggag 15600 atctatggcc ccccgaagaa ggaagagcag gattacaagc cccgaaagct aaagcgggtc 15660 aaaaagaaaa agaaagatga tgatgatgaa cttgacgacg aggtggaact gctgcacgct 15720 accgcgccca ggcgacgggt acagtggaaa ggtcgacgcg taaaacgtgt tttgcgaccc 15780 ggcaccaccg tagtctttac gcccggtgag cgctccaccc gcacctacaa gcgcgtgtat 15840 gatgaggtgt acggcgacga ggacctgctt gagcaggcca acgagcgcct cggggagttt 15900 gcctacggaa agcggcataa ggacatgctg gcgttgccgc tggacgaggg caacccaaca 15960 cctagcctaa agcccgtaac actgcagcag gtgctgcccg cgcttgcacc gtccgaagaa 16020 aagcgcggcc taaagcgcga gtctggtgac ttggcaccca ccgtgcagct gatggtaccc 16080 aagcgccagc gactggaaga tgtcttggaa aaaatgaccg tggaacctgg gctggagccc 16140 gaggtccgcg tgcggccaat caagcaggtg gcgccgggac tgggcgtgca gaccgtggac 16200 gttcagatac ccactaccag tagcaccagt attgccaccg ccacagaggg catggagaca 16260 caaacgtccc cggttgcctc agcggtggcg gatgccgcgg tgcaggcggt cgctgcggcc 16320 gcgtccaaga cctctacgga ggtgcaaacg gacccgtgga tgtttcgcgt ttcagccccc 16380 cggcgcccgc gcggttcgag gaagtacggc gccgccagcg cgctactgcc cgaatatgcc 16440 ctacatcctt ccattgcgcc tacccccggc tatcgtggct acacctaccg ccccagaaga 16500 cgagcaacta cccgacgccg aaccaccact ggaacccgcc gccgccgtcg ccgtcgccag 16560 cccgtgctgg ccccgatttc cgtgcgcagg gtggctcgcg aaggaggcag gaccctggtg 16620 ctgccaacag cgcgctacca ccccagcatc gtttaaaagc cggtctttgt ggttcttgca 16680 gatatggccc tcacctgccg cctccgtttc ccggtgccgg gattccgagg aagaatgcac 16740 cgtaggaggg gcatggccgg ccacggcctg acgggcggca tgcgtcgtgc gcaccaccgg 16800 cggcggcgcg cgtcgcaccg tcgcatgcgc ggcggtatcc tgcccctcct tattccactg 16860 atcgccgcgg cgattggcgc cgtgcccgga attgcatccg tggccttgca ggcgcagaga 16920 cactgattaa aaacaagttg catgtggaaa aatcaaaata aaaagtctgg actctcacgc 16980 tcgcttggtc ctgtaactat tttgtagaat ggaagacatc aactttgcgt ctctggcccc 17040 gcgacacggc tcgcgcccgt tcatgggaaa ctggcaagat atcggcacca gcaatatgag 17100 cggtggcgcc ttcagctggg gctcgctgtg gagcggcatt aaaaatttcg gttccaccgt 17160 taagaactat ggcagcaagg cctggaacag cagcacaggc cagatgctga gggataagtt 17220 gaaagagcaa aatttccaac aaaaggtggt agatggcctg gcctctggca ttagcggggt 17280 ggtggacctg gccaaccagg cagtgcaaaa taagattaac agtaagcttg atccccgccc 17340 tcccgtagag gagcctccac cggccgtgga gacagtgtct ccagaggggc gtggcgaaaa 17400 gcgtccgcgc cccgacaggg aagaaactct ggtgacgcaa atagacgagc ctccctcgta 17460 cgaggaggca ctaaagcaag gcctgcccac cacccgtccc atcgcgccca tggctaccgg 17520 agtgctgggc cagcacacac ccgtaacgct ggacctgcct ccccccgccg acacccagca 17580 gaaacctgtg ctgccaggcc cgaccgccgt tgttgtaacc cgtcctagcc gcgcgtccct 17640 gcgccgcgcc gccagcggtc cgcgatcgtt gcggcccgta gccagtggca actggcaaag 17700 cacactgaac agcatcgtgg gtctgggggt gcaatccctg aagcgccgac gatgcttctg 17760 aatagctaac gtgtcgtatg tgtgtcatgt atgcgtccat gtcgccgcca gaggagctgc 17820 tgagccgccg cgcgcccgct ttccaagatg gctacccctt cgatgatgcc gcagtggtct 17880 tacatgcaca tctcgggcca ggacgcctcg gagtacctga gccccgggct ggtgcagttt 17940 gcccgcgcca ccgagacgta cttcagcctg aataacaagt ttagaaaccc cacggtggcg 18000 cctacgcacg acgtgaccac agaccggtcc cagcgtttga cgctgcggtt catccctgtg 18060 gaccgtgagg atactgcgta ctcgtacaag gcgcggttca ccctagctgt gggtgataac 18120 cgtgtgctgg acatggcttc cacgtacttt gacatccgcg gcgtgctgga caggggccct 18180 acttttaagc cctactctgg cactgcctac aacgccctgg ctcccaaggg tgccccaaat 18240 ccttgcgaat gggatgaagc tgctactgct cttgaaataa acctagaaga agaggacgat 18300 gacaacgaag acgaagtaga cgagcaagct gagcagcaaa aaactcacgt atttgggcag 18360 gcgccttatt ctggtataaa tattacaaag gagggtattc aaataggtgt cgaaggtcaa 18420 acacctaaat atgccgataa aacatttcaa cctgaacctc aaataggaga atctcagtgg 18480 tacgaaactg aaattaatca tgcagctggg agagtcctta aaaagactac cccaatgaaa 18540 ccatgttacg gttcatatgc aaaacccaca aatgaaaatg gagggcaagg cattcttgta 18600 aagcaacaaa atggaaagct agaaagtcaa gtggaaatgc aatttttctc aactactgag 18660 gcgaccgcag gcaatggtga taacttgact cctaaagtgg tattgtacag tgaagatgta 18720 gatatagaaa ccccagacac tcatatttct tacatgccca ctattaagga aggtaactca 18780 cgagaactaa tgggccaaca atctatgccc aacaggccta attacattgc ttttagggac 18840 aattttattg gtctaatgta ttacaacagc acgggtaata tgggtgttct ggcgggccaa 18900 gcatcgcagt tgaatgctgt tgtagatttg caagacagaa acacagagct ttcataccag 18960 cttttgcttg attccattgg tgatagaacc aggtactttt ctatgtggaa tcaggctgtt 19020 gacagctatg atccagatgt tagaattatt gaaaatcatg gaactgaaga tgaacttcca 19080 aattactgct ttccactggg aggtgtgatt aatacagaga ctcttaccaa ggtaaaacct 19140 aaaacaggtc aggaaaatgg atgggaaaaa gatgctacag aattttcaga taaaaatgaa 19200 ataagagttg gaaataattt tgccatggaa atcaatctaa atgccaacct gtggagaaat 19260 ttcctgtact ccaacatagc gctgtatttg cccgacaagc taaagtacag tccttccaac 19320 gtaaaaattt ctgataaccc aaacacctac gactacatga acaagcgagt ggtggctccc 19380 gggttagtgg actgctacat taaccttgga gcacgctggt cccttgacta tatggacaac 19440 gtcaacccat ttaaccacca ccgcaatgct ggcctgcgct accgctcaat gttgctgggc 19500 aatggtcgct atgtgccctt ccacatccag gtgcctcaga agttctttgc cattaaaaac 19560 ctccttctcc tgccgggctc atacacctac gagtggaact tcaggaagga tgttaacatg 19620 gttctgcaga gctccctagg aaatgaccta agggttgacg gagccagcat taagtttgat 19680 agcatttgcc tttacgccac cttcttcccc atggcccaca acaccgcctc cacgcttgag 19740 gccatgctta gaaacgacac caacgaccag tcctttaacg actatctctc cgccgccaac 19800 atgctctacc ctatacccgc caacgctacc aacgtgccca tatccatccc ctcccgcaac 19860 tgggcggctt tccgcggctg ggccttcacg cgccttaaga ctaaggaaac cccatcactg 19920 ggctcgggct acgaccctta ttacacctac tctggctcta taccctacct agatggaacc 19980 ttttacctca accacacctt taagaaggtg gccattacct ttgactcttc tgtcagctgg 20040 cctggcaatg accgcctgct tacccccaac gagtttgaaa ttaagcgctc agttgacggg 20100 gagggttaca acgttgccca gtgtaacatg accaaagact ggttcctggt acaaatgcta 20160 gctaactaca acattggcta ccagggcttc tatatcccag agagctacaa ggaccgcatg 20220 tactccttct ttagaaactt ccagcccatg agccgtcagg tggtggatga tactaaatac 20280 aaggactacc aacaggtggg catcctacac caacacaaca actctggatt tgttggctac 20340 cttgccccca ccatgcgcga aggacaggcc taccctgcta acttccccta tccgcttata 20400 ggcaagaccg cagttgacag cattacccag aaaaagtttc tttgcgatcg caccctttgg 20460 cgcatcccat tctccagtaa ctttatgtcc atgggcgcac tcacagacct gggccaaaac 20520 cttctctacg ccaactccgc ccacgcgcta gacatgactt ttgaggtgga tcccatggac 20580 gagcccaccc ttctttatgt tttgtttgaa gtctttgacg tggtccgtgt gcaccggccg 20640 caccgcggcg tcatcgaaac cgtgtacctg cgcacgccct tctcggccgg caacgccaca 20700 acataaagaa gcaagcaaca tcaacaacag ctgccgccat gggctccagt gagcaggaac 20760 tgaaagccat tgtcaaagat cttggttgtg ggccatattt tttgggcacc tatgacaagc 20820 gctttccagg ctttgtttct ccacacaagc tcgcctgcgc catagtcaat acggccggtc 20880 gcgagactgg gggcgtacac tggatggcct ttgcctggaa cccgcactca aaaacatgct 20940 acctctttga gccctttggc ttttctgacc agcgactcaa gcaggtttac cagtttgagt 21000 acgagtcact cctgcgccgt agcgccattg cttcttcccc cgaccgctgt ataacgctgg 21060 aaaagtccac ccaaagcgta caggggccca actcggccgc ctgtggacta ttctgctgca 21120 tgtttctcca cgcctttgcc aactggcccc aaactcccat ggatcacaac cccaccatga 21180 accttattac cggggtaccc aactccatgc tcaacagtcc ccaggtacag cccaccctgc 21240 gtcgcaacca ggaacagctc tacagcttcc tggagcgcca ctcgccctac ttccgcagcc 21300 acagtgcgca gattaggagc gccacttctt tttgtcactt gaaaaacatg taaaaataat 21360 gtactagaga cactttcaat aaaggcaaat gcttttattt gtacactctc gggtgattat 21420 ttacccccac ccttgccgtc tgcgccgttt aaaaatcaaa ggggttctgc cgcgcatcgc 21480 tatgcgccac tggcagggac acgttgcgat actggtgttt agtgctccac ttaaactcag 21540 gcacaaccat ccgcggcagc tcggtgaagt tttcactcca caggctgcgc accatcacca 21600 acgcgtttag caggtcgggc gccgatatct tgaagtcgca gttggggcct ccgccctgcg 21660 cgcgcgagtt gcgatacaca gggttgcagc actggaacac tatcagcgcc gggtggtgca 21720 cgctggccag cacgctcttg tcggagatca gatccgcgtc caggtcctcc gcgttgctca 21780 gggcgaacgg agtcaacttt ggtagctgcc ttcccaaaaa gggcgcgtgc ccaggctttg 21840 agttgcactc gcaccgtagt ggcatcaaaa ggtgaccgtg cccggtctgg gcgttaggat 21900 acagcgcctg cataaaagcc ttgatctgct taaaagccac ctgagccttt gcgccttcag 21960 agaagaacat gccgcaagac ttgccggaaa actgattggc cggacaggcc gcgtcgtgca 22020 cgcagcacct tgcgtcggtg ttggagatct gcaccacatt tcggccccac cggttcttca 22080 cgatcttggc cttgctagac tgctccttca gcgcgcgctg cccgttttcg ctcgtcacat 22140 ccatttcaat cacgtgctcc ttatttatca taatgcttcc gtgtagacac ttaagctcgc 22200 cttcgatctc agcgcagcgg tgcagccaca acgcgcagcc cgtgggctcg tgatgcttgt 22260 aggtcacctc tgcaaacgac tgcaggtacg cctgcaggaa tcgccccatc atcgtcacaa 22320 aggtcttgtt gctggtgaag gtcagctgca acccgcggtg ctcctcgttc agccaggtct 22380 tgcatacggc cgccagagct tccacttggt caggcagtag tttgaagttc gcctttagat 22440 cgttatccac gtggtacttg tccatcagcg cgcgcgcagc ctccatgccc ttctcccacg 22500 cagacacgat cggcacactc agcgggttca tcaccgtaat ttcactttcc gcttcgctgg 22560 gctcttcctc ttcctcttgc gtccgcatac cacgcgccac tgggtcgtct tcattcagcc 22620 gccgcactgt gcgcttacct cctttgccat gcttgattag caccggtggg ttgctgaaac 22680 ccaccatttg tagcgccaca tcttctcttt cttcctcgct gtccacgatt acctctggtg 22740 atggcgggcg ctcgggcttg ggagaagggc gcttcttttt cttcttgggc gcaatggcca 22800 aatccgccgc cgaggtcgat ggccgcgggc tgggtgtgcg cggcaccagc gcgtcttgtg 22860 atgagtcttc ctcgtcctcg gactcgatac gccgcctcat ccgctttttt gggggcgccc 22920 ggggaggcgg cggcgacggg gacggggacg acacgtcctc catggttggg ggacgtcgcg 22980 ccgcaccgcg tccgcgctcg ggggtggttt cgcgctgctc ctcttcccga ctggccattt 23040 ccttctccta taggcagaaa aagatcatgg agtcagtcga gaagaaggac agcctaaccg 23100 ccccctctga gttcgccacc accgcctcca ccgatgccgc caacgcgcct accaccttcc 23160 ccgtcgaggc acccccgctt gaggaggagg aagtgattat cgagcaggac ccaggttttg 23220 taagcgaaga cgacgaggac cgctcagtac caacagagga taaaaagcaa gaccaggaca 23280 acgcagaggc aaacgaggaa caagtcgggc ggggggacga aaggcatggc gactacctag 23340 atgtgggaga cgacgtgctg ttgaagcatc tgcagcgcca gtgcgccatt atctgcgacg 23400 cgttgcaaga gcgcagcgat gtgcccctcg ccatagcgga tgtcagcctt gcctacgaac 23460 gccacctatt ctcaccgcgc gtacccccca aacgccaaga aaacggcaca tgcgagccca 23520 acccgcgcct caacttctac cccgtatttg ccgtgccaga ggtgcttgcc acctatcaca 23580 tctttttcca aaactgcaag atacccctat cctgccgtgc caaccgcagc cgagcggaca 23640 agcagctggc cttgcggcag ggcgctgtca tacctgatat cgcctcgctc aacgaagtgc 23700 caaaaatctt tgagggtctt ggacgcgacg agaagcgcgc ggcaaacgct ctgcaacagg 23760 aaaacagcga aaatgaaagt cactctggag tgttggtgga actcgagggt gacaacgcgc 23820 gcctagccgt actaaaacgc agcatcgagg tcacccactt tgcctacccg gcacttaacc 23880 taccccccaa ggtcatgagc acagtcatga gtgagctgat cgtgcgccgt gcgcagcccc 23940 tggagaggga tgcaaatttg caagaacaaa cagaggaggg cctacccgca gttggcgacg 24000 agcagctagc gcgctggctt caaacgcgcg agcctgccga cttggaggag cgacgcaaac 24060 taatgatggc cgcagtgctc gttaccgtgg agcttgagtg catgcagcgg ttctttgctg 24120 acccggagat gcagcgcaag ctagaggaaa cattgcacta cacctttcga cagggctacg 24180 tacgccaggc ctgcaagatc tccaacgtgg agctctgcaa cctggtctcc taccttggaa 24240 ttttgcacga aaaccgcctt gggcaaaacg tgcttcattc cacgctcaag ggcgaggcgc 24300 gccgcgacta cgtccgcgac tgcgtttact tatttctatg ctacacctgg cagacggcca 24360 tgggcgtttg gcagcagtgc ttggaggagt gcaacctcaa ggagctgcag aaactgctaa 24420 agcaaaactt gaaggaccta tggacggcct tcaacgagcg ctccgtggcc gcgcacctgg 24480 cggacatcat tttccccgaa cgcctgctta aaaccctgca acagggtctg ccagacttca 24540 ccagtcaaag catgttgcag aactttagga actttatcct agagcgctca ggaatcttgc 24600 ccgccacctg ctgtgcactt cctagcgact ttgtgcccat taagtaccgc gaatgccctc 24660 cgccgctttg gggccactgc taccttctgc agctagccaa ctaccttgcc taccactctg 24720 acataatgga agacgtgagc ggtgacggtc tactggagtg tcactgtcgc tgcaacctat 24780 gcaccccgca ccgctccctg gtttgcaatt cgcagctgct taacgaaagt caaattatcg 24840 gtacctttga gctgcagggt ccctcgcctg acgaaaagtc cgcggctccg gggttgaaac 24900 tcactccggg gctgtggacg tcggcttacc ttcgcaaatt tgtacctgag gactaccacg 24960 cccacgagat taggttctac gaagaccaat cccgcccgcc aaatgcggag cttaccgcct 25020 gcgtcattac ccagggccac attcttggcc aattgcaagc catcaacaaa gcccgccaag 25080 agtttctgct acgaaaggga cggggggttt acttggaccc ccagtccggc gaggagctca 25140 acccaatccc cccgccgccg cagccctatc agcagcagcc gcgggccctt gcttcccagg 25200 atggcaccca aaaagaagct gcagctgccg ccgccaccca cggacgagga ggaatactgg 25260 gacagtcagg cagaggaggt tttggacgag gaggaggagg acatgatgga agactgggag 25320 agcctagacg aggaagcttc cgaggtcgaa gaggtgtcag acgaaacacc gtcaccctcg 25380 gtcgcattcc cctcgccggc gccccagaaa tcggcaaccg gttccagcat ggctacaacc 25440 tccgctcctc aggcgccgcc ggcactgccc gttcgccgac ccaaccgtag atgggacacc 25500 actggaacca gggccggtaa gtccaagcag ccgccgccgt tagcccaaga gcaacaacag 25560 cgccaaggct accgctcatg gcgcgggcac aagaacgcca tagttgcttg cttgcaagac 25620 tgtgggggca acatctcctt cgcccgccgc tttcttctct accatcacgg cgtggccttc 25680 ccccgtaaca tcctgcatta ctaccgtcat ctctacagcc catactgcac cggcggcagc 25740 ggcagcggca gcaacagcag cggccacaca gaagcaaagg cgaccggata gcaagactct 25800 gacaaagccc aagaaatcca cagcggcggc agcagcagga ggaggagcgc tgcgtctggc 25860 gcccaacgaa cccgtatcga cccgcgagct tagaaacagg atttttccca ctctgtatgc 25920 tatatttcaa cagagcaggg gccaagaaca agagctgaaa ataaaaaaca ggtctctgcg 25980 atccctcacc cgcagctgcc tgtatcacaa aagcgaagat cagcttcggc gcacgctgga 26040 agacgcggag gctctcttca gtaaatactg cgcgctgact cttaaggact agtttcgcgc 26100 cctttctcaa atttaagcgc gaaaactacg tcatctccag cggccacacc cggcgccagc 26160 acctgtcgtc agcgccatta tgagcaagga aattcccacg ccctacatgt ggagttacca 26220 gccacaaatg ggacttgcgg ctggagctgc ccaagactac tcaacccgaa taaactacat 26280 gagcgcggga ccccacatga tatcccgggt caacggaatc cgcgcccacc gaaaccgaat 26340 tctcttggaa caggcggcta ttaccaccac acctcgtaat aaccttaatc cccgtagttg 26400 gcccgctgcc ctggtgtacc aggaaagtcc cgctcccacc actgtggtac ttcccagaga 26460 cgcccaggcc gaagttcaga tgactaactc aggggcgcag cttgcgggcg gctttcgtca 26520 cagggtgcgg tcgcccgggc agggtataac tcacctgaca atcagagggc gaggtattca 26580 gctcaacgac gagtcggtga gctcctcgct tggtctccgt ccggacggga catttcagat 26640 cggcggcgcc ggccgctctt cattcacgcc tcgtcaggca atcctaactc tgcagacctc 26700 gtcctctgag ccgcgctctg gaggcattgg aactctgcaa tttattgagg agtttgtgcc 26760 atcggtctac tttaacccct tctcgggacc tcccggccac tatccggatc aatttattcc 26820 taactttgac gcggtaaagg actcggcgga cggctacgac tgaatgttaa gtggagaggc 26880 agagcaactg cgcctgaaac acctggtcca ctgtcgccgc cacaagtgct ttgcccgcga 26940 ctccggtgag ttttgctact ttgaattgcc cgaggatcat atcgagggcc cggcgcacgg 27000 cgtccggctt accgcccagg gagagcttgc ccgtagcctg attcgggagt ttacccagcg 27060 ccccctgcta gttgagcggg acaggggacc ctgtgttctc actgtgattt gcaactgtcc 27120 taaccctgga ttacatcaag atctttgttg ccatctctgt gctgagtata ataaatacag 27180 aaattaaaat atactggggc tcctatcgcc atcctgtaaa cgccaccgtc ttcacccgcc 27240 caagcaaacc aaggcgaacc ttacctggta cttttaacat ctctccctct gtgatttaca 27300 acagtttcaa cccagacgga gtgagtctac gagagaacct ctccgagctc agctactcca 27360 tcagaaaaaa caccaccctc cttacctgcc gggaacgtac gagtgcgtca ccggccgctg 27420 caccacacct accgcctgac cgtaaaccag actttttccg gacagacctc aataactctg 27480 tttaccagaa caggaggtga gcttagaaaa cccttagggt attaggccaa aggcgcagct 27540 actgtggggt ttatgaacaa ttcaagcaac tctacgggct attctaattc aggtttctct 27600 agaaatggac ggaattatta cagagcagcg cctgctagaa agacgcaggg cagcggccga 27660 gcaacagcgc atgaatcaag agctccaaga catggttaac ttgcaccagt gcaaaagggg 27720 tatcttttgt ctggtaaagc aggccaaagt cacctacgac agtaatacca ccggacaccg 27780 ccttagctac aagttgccaa ccaagcgtca gaaattggtg gtcatggtgg gagaaaagcc 27840 cattaccata actcagcact cggtagaaac cgaaggctgc attcactcac cttgtcaagg 27900 acctgaggat ctctgcaccc ttattaagac cctgtgcggt ctcaaagatc ttattccctt 27960 taactaataa aaaaaaataa taaagcatca cttacttaaa atcagttagc aaatttctgt 28020 ccagtttatt cagcagcacc tccttgccct cctcccagct ctggtattgc agcttcctcc 28080 tggctgcaaa ctttctccac aatctaaatg gaatgtcagt ttcctcctgt tcctgtccat 28140 ccgcacccac tatcttcatg ttgttgcaga tgaagcgcgc aagaccgtct gaagatacct 28200 tcaaccccgt gtatccatat gacacggaaa ccggtcctcc aactgtgcct tttcttactc 28260 ctccctttgt atcccccaat gggtttcaag agagtccccc tggggtactc tctttgcgcc 28320 tatccgaacc tctagttacc tccaatggca tgcttgcgct caaaatgggc aacggcctct 28380 ctctggacga ggccggcaac cttacctccc aaaatgtaac cactgtgagc ccacctctca 28440 aaaaaaccaa gtcaaacata aacctggaaa tatctgcacc cctcacagtt acctcagaag 28500 ccctaactgt ggctgccgcc gcacctctaa tggtcgcggg caacacactc accatgcaat 28560 cacaggcccc gctaaccgtg cacgactcca aacttagcat tgccacccaa ggacccctca 28620 cagtgtcaga aggaaagcta gccctgcaaa catcaggccc cctcaccacc accgatagca 28680 gtacccttac tatcactgcc tcaccccctc taactactgc cactggtagc ttgggcattg 28740 acttgaaaga gcccatttat acacaaaatg gaaaactagg actaaagtac ggggctcctt 28800 tgcatgtaac agacgaccta aacactttga ccgtagcaac tggtccaggt gtgactatta 28860 ataatacttc cttgcaaact aaagttactg gagccttggg ttttgattca caaggcaata 28920 tgcaacttaa tgtagcagga ggactaagga ttgattctca aaacagacgc cttatacttg 28980 atgttagtta tccgtttgat gctcaaaacc aactaaatct aagactagga cagggccctc 29040 tttttataaa ctcagcccac aacttggata ttaactacaa caaaggcctt tacttgttta 29100 cagcttcaaa caattccaaa aagcttgagg ttaacctaag cactgccaag gggttgatgt 29160 ttgacgctac agccatagcc attaatgcag gagatgggct tgaatttggt tcacctaatg 29220 caccaaacac aaatcccctc aaaacaaaaa ttggccatgg cctagaattt gattcaaaca 29280 aggctatggt tcctaaacta ggaactggcc ttagttttga cagcacaggt gccattacag 29340 taggaaacaa aaataatgat aagctaactt tgtggaccac accagctcca tctcctaact 29400 gtagactaaa tgcagagaaa gatgctaaac tcactttggt cttaacaaaa tgtggcagtc 29460 aaatacttgc tacagtttca gttttggctg ttaaaggcag tttggctcca atatctggaa 29520 cagttcaaag tgctcatctt attataagat ttgacgaaaa tggagtgcta ctaaacaatt 29580 ccttcctgga cccagaatat tggaacttta gaaatggaga tcttactgaa ggcacagcct 29640 atacaaacgc tgttggattt atgcctaacc tatcagctta tccaaaatct cacggtaaaa 29700 ctgccaaaag taacattgtc agtcaagttt acttaaacgg agacaaaact aaacctgtaa 29760 cactaaccat tacactaaac ggtacacagg aaacaggaga cacaactcca agtgcatact 29820 ctatgtcatt ttcatgggac tggtctggcc acaactacat taatgaaata tttgccacat 29880 cctcttacac tttttcatac attgcccaag aataaagaat cgtttgtgtt atgtttcaac 29940 gtgtttattt ttcaattgcc cgggatcggt gatcaccgat ccagacatga taagatacat 30000 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 30060 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttcccggatc 30120 gcgatccggc ccgaggctgt agccgacgat ggtgcgccag gagagttgtt gattcattgt 30180 ttgcctccct gctgcggttt ttcaccgaag ttcatgccag tccagcgttt ttgcagcaga 30240 aaagccgccg acttcggttt gcggtcgcga gtgaagatcc ctttcttgtt accgccaacg 30300 cgcaatatgc cttgcgaggt cgcaaaatcg gcgaaattcc atacctgttc accgacgacg 30360 gcgctgacgc gatcaaagac gcggtgatac atatccagcc atgcacactg atactcttca 30420 ctccacatgt cggtgtacat tgagtgcagc ccggctaacg tatccacgcc gtattcggtg 30480 atgataatcg gctgatgcag tttctcctgc caggccagaa gttctttttc cagtaccttc 30540 tctgccgttt ccaaatcgcc gctttggaca taccatccgt aataacggtt caggcacagc 30600 acatcaaaga gatcgctgat ggtatcggtg tgagcgtcgc agaacattac attgacgcag 30660 gtgatcggac gcgtcgggtc gagtttacgc gttgcttccg ccagtggcgc gaaatattcc 30720 cgtgcacctt gcggacgggt atccggttcg ttggcaatac tccacatcac cacgcttggg 30780 tggtttttgt cacgcgctat cagctcttta atcgcctgta agtgcgcttg ctgagtttcc 30840 ccgttgactg cctcttcgct gtacagttct ttcggcttgt tgcccgcttc gaaaccaatg 30900 cctaaagaga ggttaaagcc gacagcagca gtttcatcaa tcaccacgat gccatgttca 30960 tctgcccagt cgagcatctc ttcagcgtaa gggtaatgcg aggtacggta ggagttggcc 31020 ccaatccagt ccattaatgc gtggtcgtgc accatcagca cgttatcgaa tcctttgcca 31080 cgcaagtccg catcttcatg acgaccaaag ccagtaaagt agaacggttt gtggttaatc 31140 aggaactgtt cgcccttcac tgccactgac cggatgccga cgcgaagcgg gtagatatca 31200 cactctgtct ggcttttggc tgtgacgcac agttcataga gataaccttc acccggttgc 31260 cagaggtgcg gattcaccac ttgcaaagtc ccgctagtgc cttgtccagt tgcaaccacc 31320 tgttgatccg catcacgcag ttcaacgctg acatcaccat tggccaccac ctgccagtca 31380 acagacgcgt ggttacagtc ttgcgcgaca tgcgtcacca cggtgatatc gtccacccag 31440 gtgttcggcg tggtgtagag cattacgctg cgatggattc cggcatagtt aaagaaatca 31500 tggaagtaag actgcttttt cttgccgttt tcgtcggtaa tcaccattcc cggcgggata 31560 gtctgccagt tcagttcgtt gttcacacaa acggtgatac gtacactttt cccggcaata 31620 acatacggcg tgacatcggc ttcaaatggc gtatagccgc cctgatgctc catcacttcc 31680 tgattattga cccacacttt gccgtaatga gtgaccgcat cgaaacgcag cacgatacgc 31740 tggcctgccc aacctttcgg tataaagact tcgcgctgat accagacgtt gcccgcataa 31800 ttacgaatat ctgcatcggc gaactgatcg ttaaaactgc ctggcacagc aattgcccgg 31860 ctttcttgta acgcgctttc ccaccaacgc tgatcaattc cacagttttc gcgatccaga 31920 ctgaatgccc acaggccgtc gagttttttg atttcacggg ttggggtttc tacaggacgg 31980 accatgcgtt cgacctttct cttctttttt gggcccatga tggcagatcc gtatagtgag 32040 tcgtattagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc cccagcatgc 32100 ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata 32160 gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg 32220 ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg 32280 caactagaag gcacagtcga ggctgatcag cgagctctag atgcatgctc gagcggccgc 32340 cagtgtgatg gatatctgca gaattccagc acactggcgg ccgttactag tggatccgag 32400 ctcggtaccc ggccgttata acaccactcg acacggcacc agctcaatca gtcacagtgt 32460 aaaaaagggc caagtgcaga gcgagtatat ataggactaa aaaatgacgt aacggttaaa 32520 gtccacaaaa aacacccaga aaaccgcacg cgaacctacg cccagaaacg aaagccaaaa 32580 aacccacaac ttcctcaaat cgtcacttcc gttttcccac gttacgtcac ttcccatttt 32640 aagaaaacta caattcccaa cacatacaag ttactccgcc ctaaaaccta cgtcacccgc 32700 cccgttccca cgccccgcgc cacgtcacaa actccacccc ctcattatca tattggcttc 32760 aatccaaaat aaggtatatt attgatgatg ggtcgtta 32798 2 465 DNA DNA encoding tumor necrosis factor-alpha misc_feature SEQ ID NO2 represents the nucleotides of positions 1469 through 1933 of SEQ ID NO1 2 tcatcttctc gaaccccgag tgacaagcct gtagcccatg ttgtagcaaa ccctcaagct 60 gaggggcagc tccagtggct gaaccgccgg gccaatgccc tcctggccaa tggcgtggag 120 ctgagagata accagctggt ggtgccatca gagggcctgt acctcatcta ctcccaggtc 180 ctcttcaagg gccaaggctg cccctccacc catgtgctcc tcacccacac catcagccgc 240 atcgccgtct cctaccagac caaggtcaac ctcctctctg ccatcaagag cccctgccag 300 agggagaccc cagagggggc tgaggccaag ccctggtatg agcccatcta tctgggaggg 360 gtcttccagc tggagaaggg tgaccgactc agcgctgaga tcaatcggcc cgactatctc 420 gactttgccg agtctgggca ggtctacttt gggatcattg ccctg 465 

What is claimed is:
 1. An adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, and optionally deficient in the E1 region, the E2A region, and/or the E3 region of the adenoviral genome, (b) a nucleic acid sequence coding for TNF, and (c) a radiation inducible promoter operably linked to the nucleic acid sequence coding for TNF.
 2. The adenoviral vector of claim 1, wherein the adenoviral genome is deficient in the E1 region of the adenoviral genome.
 3. The adenoviral vector of claim 2, wherein the nucleic acid sequence coding for TNF is located in the E1 region of the adenoviral genome.
 4. The adenoviral vector of claim 3, wherein the TNF is TNF-α.
 5. The adenoviral vector of claim 3, wherein the nucleic acid sequence coding for TNF further comprises a transcription-terminating region.
 6. The adenoviral vector of claim 1, wherein the radiation inducible promoter is Egr-1.
 7. The adenoviral vector of claim 2, wherein the adenoviral vector is deficient in the E3 region of the adenoviral genome.
 8. The adenoviral vector of claim 7, wherein the adenoviral genome is deficient in the entire E3 region.
 9. The adenoviral vector of claim 1, wherein the adenoviral genome is deficient in the entire coding region of the E4 region.
 10. The adenoviral vector of claim 9, wherein the coding region of the E4 region of the adenoviral genome has been replaced with a spacer element having at least 15 base pairs.
 11. The adenoviral vector of claim 10, wherein the spacer element is transcriptionally inert.
 12. The adenoviral vector of claim 11, wherein the spacer element comprises a polyadenylation sequence that is non-native to the E4 region of the adenoviral genome.
 13. The adenoviral vector of claim 12, wherein the spacer element comprises an SV40 polyadenylation sequence.
 14. The adenoviral vector of claim 10, wherein the viral production level of the adenoviral vector is greater than the viral production level of the adenoviral vector without the spacer element.
 15. The adenoviral vector of claim 1, wherein the adenoviral genome is a serotype-5 adenoviral genome.
 16. A replication competent adenovirus-free stock of the adenoviral vector of claim
 1. 17. A host cell comprising the adenoviral vector of claim
 1. 18. An adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, and optionally deficient in the E1 region, the E2A region, and/or the E3 region of the adenoviral genome, (b) a nucleic acid sequence coding for a TNF which is secreted from a cell infected with the adenoviral vector, and (c) a spacer element of at least 15 base pairs in the E4 region of the adenoviral genome.
 19. The adenoviral vector of claim 18, wherein the adenoviral genome is deficient in the E1 region of the adenoviral genome.
 20. The adenoviral vector of claim 18, wherein the spacer element is transcriptionally inert.
 21. The adenoviral vector of claim 20, wherein the spacer element comprises a polyadenylation sequence that is non-native to the E4 region of the adenoviral genome.
 22. The adenoviral vector of claim 21, wherein the spacer element comprises an SV40 polyadenylation sequence.
 23. The adenoviral vector of claim 19, wherein the nucleic acid sequence coding for TNF is located in the E1 region of the adenoviral genome.
 24. The adenoviral vector of claim 23, wherein the TNF is TNF-α.
 25. The adenoviral vector of claim 23, wherein the nucleic acid sequence coding for TNF further comprises a transcription-terminating region.
 26. The adenoviral vector of claim 18, wherein the adenoviral vector is deficient in the E3 region of the adenoviral genome.
 27. The adenoviral vector of claim 26, wherein the adenoviral genome is deficient in the entire E3 region.
 28. The adenoviral vector of claim 18, wherein the adenoviral genome is deficient in the entire coding region of the E4 region.
 29. The adenoviral vector of claim 18, wherein the viral production level of the adenoviral vector is greater than the viral production level of the adenoviral vector without the spacer element.
 30. The adenoviral vector of claim 18, wherein the adenoviral genome is a serotype-5 adenoviral genome.
 31. A replication competent adenoviral-free stock of the adenoviral vector of claim
 18. 32. A host cell comprising the adenoviral vector of claim
 18. 33. A method of treating a tumor or cancer in a mammal comprising administering an anti-tumor or anti-cancer effective amount of the adenoviral vector of claim 1 directly to the tumor or cancer of the mammal.
 34. The method of claim 33, further comprising the administration of radiation to the host.
 35. The method of claim 34, wherein the radiation induces expression of the nucleic acid sequence coding for TNF to produce a therapeutic level of TNF in the host.
 36. The method of claim 35, wherein the administration of radiation comprises the use of an internal source of radiation.
 37. The method of claim 33, further comprising the administration of a TNF antagonist to the host.
 38. The method of claim 37, wherein the TNF antagonist is at least one selected from the group comprising soluble TNF receptors and anti-TNF anti-bodies.
 39. A method of treating a tumor or cancer in a mammal comprising administering an anti-tumor or anti-cancer effective amount of the adenoviral vector of claim 18 directly to the tumor or cancer of the mammal.
 40. A method of producing an adenoviral vector comprising (a) providing an adenoviral genome that is deficient in the E4 region of the adenoviral genome, (b) inserting a nucleic acid sequence coding for TNF into the adenoviral genome, and (c) inserting a radiation-inducible promoter into the adenoviral genome such that it is operably linked to the nucleic acid sequence coding for TNF.
 41. The method of claim 40, wherein the adenoviral genome comprises a spacer element having at least 15 base pairs in the E4 region of the adenoviral genome.
 42. The method of claim 40, wherein the radiation inducible promoter is Egr-1.
 43. The adenoviral vector of claim 1, wherein the nucleic acid sequence encodes a TNF which is secreted from a cell infected with the adenoviral vector.
 44. The adenoviral vector of claim 1, wherein the nucleic acid sequence coding for TNF comprises SEQ ID NO:
 2. 45. The adenoviral vector of claim 18, wherein the nucleic acid sequence coding for a TNF which is secreted from a cell infected with the adenoviral vector comprises SEQ ID NO:
 2. 46. The method of claim 33, wherein the adenoviral vector is administered to a tumor in a mammal.
 47. The method of claim 46, wherein the mammal is a human.
 48. The method of claim 39, wherein the adenoviral vector is administered to a tumor in a mammal.
 49. The method of claim 48, wherein the mammal is a human.
 50. A pharmaceutical composition comprising the adenoviral vector of claim 1 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition does not contain replication-competent adenoviruses.
 51. A pharmaceutical composition comprising the adenoviral vector of claim 18 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition does not contain replication-competent adenoviruses. 